The ubiquitous tripeptide L-glutathione (GSH) (gamma-glutamyl-cysteinyl-glycine), is a well known biological antioxidant, and in fact is believed to be the primary intracellular antioxidant for higher organisms. When oxidized, it forms a dimer (GSSG), which may be recycled in organs having glutathione reductase. Glutathione may be transported through membranes by the sodium-dependent glutamate pump. Tanuguchi, N., et al. Eds., Glutathione Centennial, Academic Press, New York (1989), expressly incorporated herein by reference.
GSH is known to function directly or indirectly in many important biological phenomena, including the synthesis of proteins and DNA, transport, enzyme activity, metabolism, and protection of cells from free-radical mediated damage. GSH is one of the primary cellular antioxidants responsible for maintaining the proper oxidation state within the body. GSH is synthesized by most cells, and is also supplied in the diet. GSH has been shown to recycle oxidized biomolecules back to their active, reduced forms.
Because of the existing mechanisms for controlling interconversion of reduced and oxidized glutathione, an alteration of the level of reduced glutathione (GSH), e.g., by administration of GSH to an organism will tend to shift the cells of the organism to a more reduced redox potential. Likewise, subjecting the organism to oxidative stress or free radicals will tend to shift the cells to a more oxidized potential. It is well known that certain cellular processes are responsive to redox potential.
Reduced glutathione (GSH) is, in the human adult, produced from oxidized glutathione (GSSG) primarily by the liver, and to a smaller extent, by the skeletal muscle, red blood cells, and white cells. About 80% of the 8-10 grams glutathione produced daily is produced by the liver and distributed through the blood stream to the other tissues.
A deficiency of glutathione in cells may lead to excess free radicals, which cause macromolecular breakdown, lipid peroxidation, buildup of toxins, and ultimately cell death. Because of the importance of glutathione in preventing this cellular oxidation, glutathione is continuously supplied to the tissues. However, under certain conditions, the normal, physiologic supplies of glutathione are insufficient, distribution inadequate or local oxidative demands too high to prevent cellular oxidation. Under certain conditions, the production of and demand for glutathione are mismatched, leading to insufficient levels on an organismal level. In other cases, certain tissues or biological processes consume glutathione so that the intracellular levels are suppressed. In either case, by increasing the serum levels of glutathione, increased amounts may be directed into the cells. In facilitated transport systems for cellular uptake, the concentration gradient which drives uptake is increased.
As with all nutrients, eating or orally ingesting the nutrient would generally be considered a desired method for increased body levels thereof. Thus, attempts at oral glutathione treatments were known, and indeed the present inventors hereof previously suggested oral glutathione administration for various indications. The protocols for administration of glutathione, however, were not optimized and therefore the bioavailability of the glutathione was unassured and variable. Prior pharmaceutical attempts by others to safely, effectively and predictably raise intracellular GSH through oral therapy with GSH have not met with demonstrated success. Experts generally believe that beneficial physiological effects of orally administered glutathione are difficult or impossible to achieve, or the efficiency is so low as to make supplementation by this route unproductive.
Because of the poor or variable results obtained, the art generally teaches that oral administration of glutathione is ineffective, forcing administration or supplementation by other routes, principally intravenously, but also by alveolar inhalation. Orally absorbed prodrugs and precursors have also been proposed or used. A known pharmacological regimen provides intravenous glutathione in combination with another agent, such as cis-platinum (a free radical associated metal drug), doxorubicin, or daunorubicin (free radical associated drugs which interact with nucleic acid metabolism), which produced toxic side effects related to free radical reactions.
The ability to harness GSH, which is a powerful, but safe substance, into an effective oral pharmaceutical had not been accomplished in the past, because of molecular instability, poor gastrointestinal absorption through existing protocols and resulting inability to reliably effect increases in intracellular GSH levels. Administering sufficient amounts to achieve physiological benefit using known oral administration protocols might lead to cysteine related kidney stones, gastric distress or flatulence.
Glutathione is relatively unstable in alkaline or oxidative environments, and is not absorbed by the stomach. It is believed that glutathione is absorbed, after oral administration, if at all, in the latter half of the duodenum and the beginning of the jejunum. It was also believed that orally administered glutathione would tend to be degraded in the stomach, and that it is particularly degraded under alkaline conditions by desulfurases and peptidases present in the duodenum. Thus, known protocols for oral administration of glutathione involved administered with meals or after eating to buffer pH extremes and dilute degradative enzymes. This protocol, however, has the effect of diluting the glutathione and delaying absorption. Studies directed at determining the oral bioavailability of glutathione under such circumstances showed poor absorption, and therefore such administration was seen as of little benefit.
Therefore, while oral dosage forms of glutathione were known, the clinical benefits of these formulations were unproved and, given the lack of predictability of their effect, these formulations were not used for the treatment of specific conditions, nor proven to have effect. Further, the known protocols for administration of glutathione did not provide convenience and high bioavailability.
The prior art thus suggests that glutathione esters might be suitable as orally bioavailable sources of glutathione, which are stable and may be rapidly absorbed. However, these are both more expensive than glutathione itself and have proven toxic.
Pure glutathione forms a flaky powder that retains a static electrical charge, due to triboelectric effects, making processing and formulation difficult. The powder particles may also have an electrostatic polarization, which is akin to an electret. Glutathione is a strong reducing agent, so that autooxidation occurs in the presence of oxygen or other oxidizing agents. U.S. Pat. No. 5,204,114, Demopoulos et al., expressly incorporated herein by reference in its entirety, provides a method of manufacturing glutathione tablets and capsules by the use of crystalline ascorbic acid as an additive to reduce triboelectric effects which interfere with high speed equipment and maintaining glutathione in a reduced state. A certain crystalline ascorbic acid is, in turn, disclosed in U.S. Pat. No. 4,454,125, Demopoulos, expressly incorporated by reference herein in its entirety. This crystalline form is useful as a lubricating agent for machinery. Ascorbic acid has the advantage that it is well tolerated, antioxidant, and reduces the net static charge on the glutathione.
In synthesizing glutathione in the body, cysteine, a thiol amino acid is required. Since the prior art suggests that oral administration of glutathione itself would be ineffective, prodrugs or precursor therapy was advocated. Therefore, the prior art suggests administration of cysteine, or a more bioavailable precursor of cysteine, N-acetyl cysteine (NAC). While cysteine and NAC are both, themselves, antioxidants, their presence competes with glutathione for resources in certain reducing (GSH recycling) pathways. Since glutathione is a specific substrate for many reducing pathways, the loading of a host with cysteine or NAC may result in less efficient utilization or recycling of glutathione. Thus, cysteine and NAC are not ideal GSH prodrugs. NAC has also demonstrated some neurotoxicity. Thus, while GSH may be degraded, transported as amino acids, and resynthesized in the cell, there may also be circumstances where GSH is transported into cells without degradation; and in fact the administration of cysteine or cysteine precursors may interfere with this process.
A number of disease states have been specifically associated with reductions in glutathione levels. Depressed glutathione levels, either locally in particular organs, or systemically, have been associated with a number of clinically defined diseases and disease states. These include HIV/AIDS, diabetes and macular degeneration, all of which progress because of excessive free radical reactions and insufficient GSH. Other chronic conditions may also be associated with GSH deficiency, including heart failure and coronary artery restenosis post angioplasty.
For example, diabetes afflicts 8% of the United States population and consumes nearly 15% of all United States healthcare costs. HIV/AIDS has infected nearly 1 million Americans. Current therapies cost in excess of $20,000 per year per patient, and are rejected by, or fail in 25% to 40% of all patients. Macular degeneration presently is considered incurable, and will afflict 15 million Americans by 2002.
Clinical and pre-clinical studies have demonstrated the linkage between a range of free radical disorders and insufficient GSH levels. Newly published data implies that diabetic complications are the result of hyperglycemic episodes that promote glycation of cellular enzymes and thereby inactivate GSH synthetic pathways. The result is GSH deficiency in diabetics, which may explain the prevalence of cataracts, hypertension, occlusive atherosclerosis, and susceptibility to infections in these patients.
GSH functions as a detoxicant by forming GSH S-conjugates with carcinogenic electrophiles, preventing reaction with DNA, and chelation complexes with heavy metals such as nickel, lead, cadmium, mercury, vanadium, and manganese. GSH also plays a role in metabolism of various drugs, such as opiates. It has been used as an adjunct therapy to treatment with nephrotoxic chemotherapeutic agents such as cisplatin, and has been reported to prevent doxorubicin-induced cardiomyopathy. GSH is also an important factor in the detoxification of acetaminophen and ethanol, two powerful hepatotoxins. See:
Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on the decomposition of glutathione. I. Decomposition in solid state. Chem. Pharm. Bull. 26: 2081-91, 1978.
Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on decomposition of glutathione. II. Anaerobic decomposition in aqueous solution. Chem. Pharm. Bull. 28: 514-20, 1980.
Aruga, M., Awazu, S. and Hanano, M.: Kinetic studies on decomposition of glutathione. III. Peptide bond cleavage and desulfurization in aqueous solution. Chem. Pharm. Bull. 28: 521-28, 1980.
Hagen, T. M., Aw, T. Y., and Jones, D. P.: Glutathione uptake and protection against oxidative injury in isolated kidney cells. Kidney Intl. 34: 74-81, 1988.
Lash, L. H., and Jones, D. P.: Distribution of oxidized and reduced forms of glutathione and cysteine in rat plasma. Arch. Biochem. Biophys. 240: 583-92, 1985.
Meister, A.: Selective modification of glutathione metabolism. Science 220: 472-477, 1983.
Meister, A. and Anderson, M. E.: Glutathione. Ann. Rev. Biochem. 52: 711-60, 1983.
Riley, R. J., Spielberg, S. P., Leeder, J. S.: A comparative study of the toxicity of chemically reactive xenobiotics towards adherent cell cultures: selective attenuation of menadione toxicity by buthionine sulphoximine pretreatment. J. Pharmacol. 45 (4): 263-267, 1993.
Wierzbicka, G. T., Hagen, T. M. & Jones, D. P.: Glutathione in food. J. Food Comp. Anal. 2: 327-337, 1989.
Bravenboer, B., Kappelle, A. C., Hamers, F. P., van Buren, T., Erkelens, D. W. & Gispen, W. H.: Potential use of glutathione for the prevention and treatment of diabetic neuropathy in the streptozocin-induced diabetic rat. Diabetologia 35: 813-817, 1992.
Cavaletti, E., Tofanetti, O. & Zunino F.: Comparison of reduced glutathione with 2-mercaptoethane sulfonate to prevent cyclophosphamide-induced urotoxicity. Cancer Letters 32: 1, 1986.
Hamers, F. P., Brakkee, J. H., Cavalletti, E., Tedeschi, M., Marmonti, L., Pezzoni, G., Neijt, J. P. & Gispen, W. H.: Reduced glutathione protects against cisplatin-induced neurotoxicity in rats. Cancer Res. 53: 544-549, 1993.
Kromidas, L., Trombetta, L. D., and Jamall, I. S.: The protective effects of glulathione against methylmercury cytotoxicity. Toxicol. Letters 51: 67-80, 1990.
Novi, A. M., Flohe, R., and Stukenkemper, S.: Glutathione and aflatoxin B1-induced liver tumors: requirement for an intact glutathione molecule for regression of malignancy in neoplastic tissue. Ann. NY Acad. Sci. 397: 62-71, 1982.
Rao, R. D. N., Fischer, V., and Mason, R. P.: Glutathione and ascorbate reduction of the acetaminophen radical formed by peroxidase. J. Biol. Chem. 265: 844-7, 1990.
Skoulis, N. P., James, R. C., Harbison, R. D. and Roberts, S. M.: Depression of hepatic glutathione by opioid analgesic drugs in mice. Toxicol. Appl. Pharmacol. 99: 139-47, 1989.
Villani, F., Galimberti, M., Zunino, F., Monti, E., Rozza, A., Favalli, L. & Poggi, P.: Prevention of doxorubicin-induced cardlomyopathy by reduced glutathione. Cancer Chemother. Pharmacol. 28: 365-369, 1991.
Wagner, G., Frenzel, H., Wefers, H. and Sies, H.: Lack of effect of long-term glutathione administration on aflatoxin B1-induced hepatoma in male rats. Chem. Biol. Interactions 53: 57-68, 1985.
Yoda, Y., Nakazawa, M., Abe, T. & Kawakami, Z.: Prevention of Doxorubicin myocardial toxicity in mice by reduced glutathione. Cancer Research 46: 2551, 1986.
Younes, M., and Strubelt, O.: Protection by exogenous glutathione against hypoxic and cyanide-induced damage to isolated perfused rat livers. Toxicol. Letters 50: 229-236, 1990.
McCartney, M. A.: Effect of glutathione depletion on morphine toxicity in mice. Biochem. Pharmacol. 38: 207-9, 1989.
Ishida, T., Kumagai, Y., Ikeda, Y., Ito, K., Yano, M., Toki, S., Mihashi, K., Fujioka, T., Iwase, Y. and Hachiyama, S.: (8S)-(glutathion-S-YL)dihydromorphinone, a novel metabolite kof morphine from guinea pig bile. Drug. Metab. Dispos. 17: 77-81, 1989.
Nagamatsu, K., Kido, Y., Teroa, T, Ishida, T. and Toki, S.: Protective effect of sulfhydryl compounds on acute toxicity of morphinone. Life Sci. 30: 1121-27, 1982.
(1) HIV
High GSH levels have been demonstrated to be necessary for proper functioning of platelets, vascular endothelial cells, macrophages, cytotoxic T-lymphocytes, and other immune system components. Recently it has been discovered that HIV-infected patients exhibit low GSH levels in plasma, in other fluids, and in certain cell types like macrophages, which does not appear to be due to defects in GSH synthesis.
Droge, W., Pottmeyer-Gerber, C., Schmidt, H. & Nick, S.: Glutathione augments the activation of cytotoxic T lymphocytes in vivo. Immunobiol. 172: 151-156, 1986.
Droge, W., Eck, H. P., Gmunder, H., and Mihm, S.: Modulation of lymphocyte functions and immune responses by cysteine and cysteine derivatives. Amer. J. Medicine 91 (3C): 140S-144S, 1991.
Furukawa, T., Meydani, S. N. & Blumberg, J. B.: Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice. Mech. Ageing Dev. 38: 107-117, 1987.
Franklin, R. A., Yong, M. L., Arkins, S., and Kelley, K. W.: Glutathione augments in vitro proliferative responses of lymphocytes to concanavalin A to a greater degree in old than in young rats. J. Nutr. 120: 1710-17, 1990.
Kavanaugh, T. J., Grossman, A., Jaecks, E. P, Jinneman, J. C., Eaton, D. L., Masrtin, G. M., and Rabinovitch, P. S.: Proliferative capacity of human peripheral lymphocytes sorted on the basis of glutathione content. J. Cell. Physiol. 145: 472-80, 1990.
Robinson, M. K, Rodrick, M. L., Jacobs, D. O., Rounds, J. D., Collins, K. H., Saproschetz, I. B., Mannick, J. A., and Wilmore, D. W.: Glutathione depletion in rats impairs T-cell and macrophage immune function. Arch. Surg. 128: 29-35, 1993.
Suthanthiran, M., Anderson, M. E., Sharma, V. K. & Meister, A.: Glutathione regulates activation-dependent DNA synthesis in highly purified normal human T lymphocytes stimulated via the CD2 and CD3 antigens. Proc. Natl. Acad. Sci. USA 87: 3343-3347, 1990.
GSH has been shown to inhibit HIV replication in chronically-infected cells and in cells acutely infected in vitro. This makes GSH replacement therapy attractive, because it has the potential to interfere with the expression of the integrated HIV genome, a site that is not attacked by the currently employed antiretrovirals (AZT, ddI, ddC, D4T). GSH may also have benefits in countering the excess free radical reactions in HIV infection, which may be attributable to: 1) the hypersecretion of TNF-.alpha. by B-lymphocytes, in HIV infection, and 2) the catalysis of arachidonic acid metabolism by the GP-120 protein of HIV. The physiologic requirements for GSH by key cell types of the immune system, and the ability of macrophages to take up intercellular GSH, as well as to metabolically interact with T-lymphocytes to indirectly cause their GSH to increase, offer additional reasons to attempt to correct the GSH deficiency in HIV/AIDS.
In other new data dealing with HIV infections, the March 1997 issue of the Proceedings of the National Academy of Sciences (PNAS) established " . . . GSH deficiency as a key determinant of survival in HIV disease . . . " GSH deficiency is associated with impaired survival in HIV disease (PNAS. Vol. 94, pp. 1967-1972). The quest to raise GSH levels in cells is widely recognized as being extremely important in HIV/AIDS and other disorders, because the low cellular GSH levels in these disease processes permit more and more free radical reactions to propel the disorders.
HIV is known to start pathologic free radical reactions that lead to the destruction of GSH, as well as exhaustion of other antioxidant systems and destruction of cellular organelles and macromolecules. In pre-clinical studies, GSH stops the replication of the virus at a unique point, and specifically prevents the production of toxic free radicals, prostaglandins, TNF-.alpha., interleukins, and a spectrum of oxidized lipids and proteins that are immunosuppressive, cause muscle wasting and neurologic symptoms. Restoring GSH levels could slow or stop the diseases progression, safely and economically.
In mammalian cells, oxidative stresses, i.e., low intracellular levels of reduced GSH, and relatively high levels of free radicals, activate certain cytokines, including NF-.kappa.B and TNF-.alpha., which, in turn, activate cellular transcription of the DNA to mRNA, resulting in translation of the mRNA To A Polypeptide Sequence. See, Sonia Schoonbroodt, Sylvie Legrand-Poels, Martin Best-Belpomme and Jacques Piette; Activation of the NF-.kappa.B transcription factor in a T-lymphocytic cell line by hypochlorous acid, Biochem. J. (1997) 321, 777-785, Flohe, L., Brigelius-Flohe, R., Saliou, C., Traber, M. G. and Packer, L., Redox regulation of NF-kappa B activation. (1997) Free Radical Biology and Medicine, 22: 1115-1126. Antioxidants have been shown to block the induction of NF-.kappa.B by oxidant agents. In a virus-infected cell, the viral genome is transcribed, resulting in viral RNA production, generally necessary for viral replication of RNA viruses and retroviruses. These processes require a relatively oxidized state of the cell, a condition which results from stress, low glutathione levels, or the production of reduced cellular products. The mechanism that activates cellular transcription is evolutionarily highly conserved, and therefore it is unlikely that a set of mutations would escape this process, or that an organism in which mutated enzyme and receptor gene products in this pathway would be well adapted for survival. Thus, by maintaining a relatively reduced state of the cell (relatively reduced redox potential), viral transcription, a necessary step in late stage viral replication, is impeded.
The amplification effect of oxidative intracellular conditions on viral replication is compounded by the actions of various viruses and viral products that degrade GSH. For example, GP-120, an HIV surface glycoprotein having a large number of disulfide bonds, and normally present on the surface of infected cells, oxidizes GSH, resulting in reduced intracellular GSH levels. On the other hand, GSH reduces disulfide bonds of GP-120, decreasing or eliminating its biological activity, which in turn is necessary for viral infectivity. GSH therefore interferes with the production of such oxidized proteins, and degrades them once formed. GSH also participates in the destruction of hydrogen peroxide, which is a long-lived oxidative messenger which has been implicated in activating NF-.kappa.B. R. Schreck, P. Rieber & P. A. Baeuerle; Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1, EMBO J 10: 2247-2258 (1991).
In a cell which is actively replicating viral gene products, a cascade of events may occur which allow the cell to pass from a relatively quiescent stage with low viral activity to an active stage with massive viral replication and cell death, accompanied by a change in cellular redox potential; by maintaining adequate GSH levels, this cascade may be impeded.
Thus, certain viral infections, such as HIV, are associated with reduced GSH levels, and it is believed that by increasing intracellular GSH levels in infected cells, as well as increasing extracellular GSH, the replication of HIV may be interfered with, and the cascade of events delayed or halted. It is noted that AIDS may also be associated with reduced GSSG levels, implying an interference with de novo synthesis of GSH as well as the oxidation of existing GSH discussed above.
Initially after infection with HIV, there is an intense viral infection simulating a severe case of the flu, with massive replication of the virus. This acute phase passes within weeks, spontaneously, as the body mounts a largely successful immune defense. Thereafter, the individual has no outward manifestations of the infection. However, the virus continues to replicate, insidiously, within immune system tissues and cells, like lymph nodes, lymphoid nodules and special multidendritic cells that are found in various body cavities.
This infection is not just a viral problem. The virus, in addition to replicating, causes excessive production of various free radicals and various cytokines in toxic or elevated levels. The latter are normally occurring biochemical substances that signal numerous reactions, usually existing in minuscule concentrations. Eventually, after an average of 7-10 years of seemingly quiescent HIV infection, the corrosive free radicals and the toxic levels of cytokines begin to cause symptoms, and failures in the immune system begin. Toxic factors, such as 15-HPETE, which is immunosuppressive, and TNF-.alpha., which causes muscle wasting, are produced. The numbers of viral particles increase and the patient develops the Acquired Immune Deficiency Syndrome, AIDS, which may last 2 to 4 years before the individual's demise. AIDS, therefore, is not simply a virus infection, although the viral infection is believed to be an integral part of the etiology of the disease.
HIV has a powerful ability to mutate. It is this capability that makes it difficult to create a vaccine or to develop long-term anti-viral pharmaceutical treatments. As more people continue to fail the present complex pharmaceutical regimens, the number of resistant viral strains is increasing. This is a particularly dangerous pool of HIV and poses a considerable threat. These resistant mutants also add to the difficulties in developing vaccines. This epidemic infection is out of control, and the widely popularized polypharmaceutical regimens that are aimed only at lowering the number of viruses are proving to be too complex, too toxic, too costly, and too narrow. As a result, since the introduction of protease inhibitors, in combination with AZT-type drugs, increasing numbers of people are failing such therapies. Further, the continuing production of free radicals and cytokines, which may become largely independent of the virus, perpetuates the dysfunctions of the immune system, the gastrointestinal tract, the nervous system, and many other organs in AIDS. The published scientific literature indicates that many of these diverse organ system dysfunctions are due to systemic GSH deficiencies that are engendered by the virus and its free radicals. GSH is consumed in HIV infections because it is the principal, bulwark antioxidant versus free radicals. An additional cause of erosion of GSH levels is the presence of numerous disulfide bonds (--S--S--) in HIV proteins, such as the GP-120 discussed above. Disulfide bonds react with GSH and oxidize it.
The current HIV/AIDS pharmaceuticals take good advantage of the concept of pharmaceutical synergism, wherein two different targets in one process are hit simultaneously. The effect is more than additive. The drugs now in use were selected to inhibit two very different points in the long path of viral replication. The pathway of viral replication can be depicted simply:
HIV Replication Pathway - - - .fwdarw. - - - .fwdarw. - - - .fwdarw. - - - .fwdarw. - - - .fwdarw. point #1 point #2 point #3 point #4 point #5 Virus attacks Virus makes Viral DNA Proviral DNA Viral RNA is and enters DNA from is integrated is inactive for produced, the cell its RNA into cells' a long time, but along with DNA activators will viral mem- start HIV branes, and replicating proteins, rapidly which are assembled Viral gp120 Reverse Integrase is NF kappa B is Viral protein and transcriptase the enzyme the activator of protease CD4+ cell is the involved dormant HIV is involved receptors and enzyme DNA and others are involved glutathione involved levels must be low for activation to occur AZT, ddl, Glutathione Protease ddC Inhibitors
Point #2 was the earliest point of attack, using AZT-types of drugs, including ddI, ddC and others. These are toxic and eventually viruses become resistant to these Reverse Transcriptase inhibitors.
Point #5 is a late replication step, and this is where protease inhibitors function. The drug blocks viral protease, an enzyme that snips long protein chains to just the right length so the viral coat fits exactly around the nucleic acid core, and that proteins having different biological activities are separated. By themselves, protease inhibitors foster the rapid development of resistant, mutant strains.
By combining Reverse Transcriptase inhibitors plus protease inhibitors, synergism was obtained and the amounts of viral particles in the plasma plummeted, while the speed of the developing mutant resistant viral strains was slowed, compared to using only one type of inhibitor. The initial promise of these combination therapies or "cocktails" has been tainted by increasing numbers of failures, which are expected to rise as resistant mutants develop, albeit more slowly than the use of the drugs separately.
New therapies include additional drugs in the classes of Reverse Transcriptase inhibitors and protease inhibitors. Also, drugs are in development to block point #3, wherein the enzyme, integrase, integrates the HIV DNA into the infected cell's DNA, analogous to splicing it small length of wire into a longer wire. Vaccine development also continues, although prospects seem poor because HIV appears to be a moving target and seems to change as rapidly as a chameleon. Vaccine development is also impaired by the immune cell affinity of the virus.
Human Immunodeficiency virus-infected individuals have lowered levels of serum acid-soluble thiols and GSH in plasma, peripheral blood monocytes, and lung epithelial lining fluid. In addition, it has been shown that CD4+ and CD8+ T cells with high intracellular GSH levels are selectively lost as HIV infection progresses. This deficiency may potentiate HIV replication and accelerate disease progression, especially in individuals with increased concentrations of inflammatory cytokines because such cytokines stimulate HIV replication more efficiently in GSH-depleted cells. GSH and glutathione precursors such as N-acetyl cysteine (NAC) can inhibit cytokine-stimulated HIV expression and replication in acutely infected cells, chronically infected cells, and in normal peripheral blood mononuclear cells.
It is noted that depletion of GSH is also associated with a processes known as apoptosis, or programmed cell death. Thus, intercellular processes that artificially deplete GSH may lead to cell death, even if the underlying process itself is not lethal. See:
Arpadi, S. M., Zang, E, Muscat J. and Richie, J.: Glutathione deficiency in HIV-1-infected children with growth failure, (submitted for publication).
Baker, D. H. and Wood, R. J.: Cellular antioxidant status and human immunodeficiency virus replication. Nutr. Rev. 50: 15-8, 1992.
Baruchel, S., and Wainberg, M. A.: The role of oxidative stress in disease progression in individuals infected by the human immunodeficiency virus. J. Leukocyte Biol. 52: 111-114, 1992.
Buhl, R., Holroyd, K. J., Mastrangli, A., Cantin, A. M., Jaffe, H. A., Wells, F. B., Saltini, C. and Crystal, R. G.: Systemic glutathione deficiency in symptom-free HIV-seropositive individuals. Lancet ii: 1294-1298, 1989.
de Quay, B., Malinverni, R. and Lauterburg, B. H.: Glutathione depletion in HIV-infected patients: role of cysteine deficiency and effect of oral N-acetylcysteine. AIDS 6: 815-9, 1992.
Droge, W., Eck, H. P. and Mihm, S.: HIV-induced cysteine deficiency and T-cell dysfunction--a rationale for treatment with N-acetylcysteine. Immunol. Today 13: 211-4, 1992.
Eck, H. P., Gmunder, H., Hartmann, M., Petzoldt, D., Daniel, V. and Droge, W.: Low concentrations of acid-soluble thiol (cysteine) in the blood plasma of HIV-infected patients. Biol. Chem. Hoppe-Seyler 370: 101-108, 1989.
Fauci, A. S.: Multifactorial nature of human immunodeficiency virus disease: Implications for therapy. Science 262: 1011-1018, 1993.
Foley, P. Kazazi, F., Biti, R., Sorrell, T. C., and Cunningham, A. L.: HIV infection of monocytes inhibits the T-lymphocyte proliferative response to recall antigens via production of eicosanoids. Immunology 75: 391-97, 1992.
Hasan, V., Thomas, D., Aclami, J. et al. : Stimulation of a human T-cell clone with anti-CD3 or tumor necrosis factor induces NFkB translocation but not human immunodeficiency virus 1 enhancer-dependent transcription. Proc. Natl. ACAD. sCI. 87: 7861-65, 1990.
Ho, W. Z. and Douglas, S. D.: Glutathione and N-acetylcysteine suppression of human immunodeficiency virus replication in human monocyte/macrophages in vitro. AIDS Res. Hum. Retroviruses, 8: 1249-53, 1992.
Israel, N., Gougerot-Pocidalo, M. A., Aillet, F., and Virelizier, J. L.: Redox status of cells influences constitutive or induced NF?B translocation and HIV long terminal repeat activity in human T and monocytic cell lines. J. Immunol. 149: 3386-93, 1992.
Kobayashi, S., Hamamoto, Y., Kobayashi, N., and Yamamoto, N.: Serum level of TNFa in HIV-infected individuals. AIDS 4: 169 1990.
Kalebic, T., Kinter, A., Poli, G., Anderson, M. E., Meister, A. and Fauci, A. S.: Suppression of human immunodeficiency virus expression in chronically infected monocytic cells by glutathione, glutathione ester, and N-acetylcysteine. Proc. Natl. Acad. Sci. USA 87: 986-990, 1991.
LeGrand-Poels, S., Vaira, D., Pincemail, J., Van de Vorst, A. and Piette, J.: Activation of human immunodeficiency virus type 1 by oxidative stress. AIDS Res. Hum. Retrov. 6: 1389-97, 1990.
Mihm, S., Ennen, J., Pessara, U., Kurth, R. and Droge, W.: Inhibition of HIV-1 replication and NF-kb activity by cysteine and cysteine derivatives. AIDS 5: 497-503, 1991.
National Institutes of Health. Dr. Howard C. Greenspan. Chairman of Conference on Free Radicals and Antioxidants in HIV/AIDS, Nov. 12-13, 1993/Greenspan, H. C. The role of reactive oxygen species, antioxidants and phytopharmaceuticals in human immunodeficiency virus activity. Med-Hypotheses 40: 85-92, 1993.
Roederer, M., Raju, P. A., Staal, F. J. T., Herzenberg, L. A. and Herzenberg, L. A.: N-acetylcysteine inhibits latent HIV expression in chronically infected cells. AIDS Res. Human Retrovir. 7: (6) 563-567, 1991.
Roederer, M., Staal, F. J. T., Osada, H., Herzenberg, L. A. and Herzenberg, L. A.: CD4 and CD8 T cells with high intracellular glutathione levels are selectively lost as the HIV infection progresses. Internat. Immunol. 3: 933-37, 1991.
Roederer, M., Staal, F. J. T., Raju, P. A., Ela, S. W., Herzenberg, L. A. and Herzenberg, L. A.: Cytokine-stimulated human immunodeficiency virus replication is inhibited by N-acetyl-L-cysteine. Proc. Natl. Acad. Sci. USA 87: 4884-4888, 1990.
Schreck, R. Rieber, P., and Baeurle, P. A.: Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kb transcription factor and HIV-1. EMBO J. 10: 2247-2258, 1991.
Staal, F. J., Roederer, M., Herzenberg, L. A. and Herzenberg, L. A.: Glutathione and immunophenotypes of T and B lymphocytes in HIV-infected individuals. Ann. NY Acad. Sci. 651: 453-63, 1992.
Staal, F. J. T., Roederer, M. Herzenberg, L. A., and Herzenberg, L. A.: Intracellular thiols regulate activation of nuclear factor kappa-B and transcription of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 87: 9943-9947, 1990.
Staal, F. J., Ela, S. W., Roederer, M., Anderson, M. T., Herzenberg, L. A. and Herzenberg, L. A.: Glutathione deficiency and human immunodeficiency virus infection. Lancet 339: 909-12, 1992.
Staal, F. J., Roederer, M., Israelski, D. M., Bubp, J., Mole, L. A., McShane, D., Deresinski, S. C., Ross, W., Sussman, H., Raju, P. A., Herzenberg, L. A. and Herzenberg, L. A.: Intracellular glutathione levels in T cell subsets decrease in HIV-infected individuals. AIDS Res. Hum. Retroviruses 8: 305-11, 1992.
Staal, F. J. T., Roederer, M., Raju, P. A., Anderson, M. T., Ela, S. W., Herzenberg, L. A., and Herzenberg, L. A.: Antioxidants inhibit stimulation of HIV transcription. AIDS Res. Hum. Retrov. 9: 299-306, 1993.
Wahl, L. M., Corcoran, M. L., Pyle, S. W., Arthur, L. O., Harel-Bellan, A. and Farrar, W. L.: Human immunodeficiency virus glycoprotein (gp120) induction of monocyte arachidonic acid metabolites and interleukin 1. Proc. Natl. Acad. Sci. 86: 621-625, 1989.
2) Diabetes Mellitus
Diabetes mellitus is found in two forms, childhood or autoimmune (type I, IDDM) and late-onset or non-insulin dependent (type II, NIDDM). The former constitute about 30% and the remainder represent the bulk of cases seen. Onset is generally sudden for Type I, and insidious for Type II. Symptoms include excessive urination, hunger and thirst with a slow steady loss of weight in the first form. Obesity is often associated with the second form and has been thought to be a causal factor in susceptible individuals. Blood sugar is often high and there is frequent spilling of sugar in the urine. If the condition goes untreated, the victim may develop ketoacidosis with a foul-smelling breath similar to someone who has been drinking alcohol. The immediate medical complications of untreated diabetes can include nervous system symptoms, and even diabetic coma.
Because of the continuous and pernicious occurrence of hyperglucosemia (very high blood sugar levels), a non-enzymatic chemical reaction occurs called glycation. Since glycation occurs far more frequently inside cells, the inactivation of essential enzyme proteins happens almost continually. One of the most critical enzymes, .gamma.-glutamyl-cysteine synthetase, is glycated and readily inactivated. This enzyme is the crucial step in the biosynthesis of glutathione in the liver.
The net result of this particular glycation is a deficiency in the production of GSH in diabetics. Normally, adults produce 8-10 grams every 24 hours, and it is rapidly oxidized by the cells. GSH is in high demand throughout the body for multiple, essential functions, for example, within all mitochondria, to produce chemical energy called ATP. Brain cells, heart cells, and others simply will not function well and can be destroyed through apoptosis.
GSH is the major antioxidant in the human body and the only one we are able to synthesize, de novo. It is also the most common small molecular weight thiol in both plants and animals. Without GSH, the immune system cannot function, and the central and peripheral nervous systems become aberrant and then cease to function. Because of the dependence on GSH as the carrier of nitric oxide, a vasodilator responsible for control of vascular tone, the cardiovascular system does not function well and eventually fails. Since all epithelial cells seem to require GSH, the intestinal lining cells don't function properly and valuable micronutrients are lost, nutrition is compromised, and microbes are given portals of entry to cause infections.
The use of GSH precursors cannot help to control the GSH deficiency due to the destruction of the rate-limiting enzyme by glycation. As GSH deficiency becomes more profound, the well-known sequellae of diabetes progress in severity. The complications described below are essentially due to runaway free radical damage since the available GSH supplies in diabetics are insufficient.
Ceriello, A., Giugliano, D., Quatraro, A. & Lefebvre, P. J.: Anti-oxidants show an anti-hypertensive effect in diabetic and hypertensive subjects. Clin. Sci. 81: 739-742, 1991.
Paolisso, G., Giugliano, D., Pizza, G., Gambardella, A., Tesauro, P., Varricchio, M. & D'Onofrio, F.: Glutathione infusion potentiates glucose-induced insulin secretion in aged patients with impaired glucose tolerance. Diabetes Care 15: 1-7, 1992.
Reducing sugars are known to interact with free amino groups in proteins, lipids, and nucleic acids to form Amadori product and produce reactive oxygen species through the glycation reaction. Under diabetic conditions, glucose level is elevated and the glycated proteins increased. Cu,Zn-SOD has been shown to be glycated and inactivated under diabetic conditions and that ROS produced from the Amadori product caused site-specific fragmentation of Cu,Zn-SOD. Fructose, which is produced through polyol pathway, has stronger glycating capacity than glucose because the physiologic proportion of the linear form is higher than that of cyclized form. Fructose, as well as ribose, can bring about apoptosis in pancreatic .beta. islet cell line. Levels of intracellular peroxides, protein carbonyls, and malondialdehyde are increased in the presence of fructose. In addition, methylglyoxal and 3-deoxyglucosone have also been shown to induce apoptotic cell death. 3-Deoxyglucosone, a 2-oxoaldehyde, is produced through the degradation of Amadori compounds. Both compounds are elevated during hyperglycemia and accelerate the glycation reaction. These compounds are toxic to cells, due to their high reactivity, and a scavenging system with NADPH-dependent reducing activity exists, including aldehyde reductase. Junichi Fujii and Naoyuki Taniguchi, Dysfunction of Redox System by Reactive Oxygen Species, Nitric Oxide and the Glycation Reaction: A Possible Mechanism for Apoptotic Cell Death (Poster), Proceedings of 3rd Internet World Congress on Biomedical Sciences, 1996, 12, 9-20 Riken, Tsukuba, Japan. See, also:
Boldin M P, Goncharov T M, Goltsev Y V, Wallach D. 1996. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death. Cell 85: 803-815.
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Takahashi M, Lu Y, Myint T, Fujii J, Wada Y, et al. 1995. In vivo glycation of aldehyde reductase, a major 3-deoxyglucosone reducing enzyme. Identification of glycation sites. Biochemistry 34: 1433-1438.
Takahashi M, Fujii J, Miyoshi E, Hoshi A, Taniguchi N. 1996. Elevation of aldose reductase gene expression in rat primary hepatoma and hepatoma cell lines: Implication in detoxification of cytotoxic aldehydes. Int. J. Cancer. 87: 337-341.
Seo H G, Takata I, Nakamura M, Tatsumi H, Suzuki K, et al. 1995. Induction of nitric oxide synthase and concomitant suppression of superoxide dismutases in experimental colitis in rats. Arch. Biochem. Biophys. 324: 41-47.
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Cell-cell adhesion is critical in generation of effective immune responses and is dependent upon the expression of a variety of cell surface receptors. Intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule (VCAM-1: CD 106) are inducible cell surface glycoproteins. The expression of these surface proteins are known to be induced in response to activators such as cytokines (TNF-.alpha., IL-1 .alpha. & .beta.), PMA, lipopolysaccharide and oxidants. The ligands for ICAM-1 and VCAM-1 on lymphocyte are LFA-1 (CD11a/CD18) and VLA-4, respectively. The inappropriate or abnormal sequestration of leukocytes at specific sites is a central component in the development of a variety of autoimmune diseases and pathologic inflammatory disorders. Focal expression of ICAM-1 have been reported in arterial endothelium overlying early foam cell lesions in both dietary and genetic models of atherosclerosis in rabbits. A role of VCAM-1 in the progression of coronary lesions has also been suggested. Loss or gain of cell surface molecules is thought to determine the mobilization, emigration and invasiveness of epithelial cancer cells. Monocytes from patients with diabetes mellitus are known to have increased adhesion to endothelial cells in culture. Regulation of adhesion molecule expression and function by reactive oxygen species via specific redox sensitive mechanisms have been reported. Antioxidants can block induced adhesion molecule expression and cell-cell adhesion. Sashwati Roy and Chandan K. Sen. Adhesion Molecules And Cell-Cell Adhesion, http://packer.berkeley.edu/research/Cell/adhes.
The diabetic will become more susceptible to infections because the immune system approaches collapse when GSH levels fall, analogous to certain defects seen in HIV/AIDS. Peripheral vasculature becomes compromised and blood supply to the extremities is severely diminished because GSH is not available in sufficient amounts to stabilize the nitric oxide (.NO) to effectively exert its vascular dilation (relaxation) property. Gangrene is a common sequel and successive amputations are often the result in later years.
Peripheral neuropathies, the loss of sensation commonly of the feet and lower extremities develop, often followed by aberrant sensations like burning or itching, which can't be controlled. Retinopathy and nephropathy are later events that are actually due to microangiopathy, excessive budding and growth of new blood vessels and capillaries, which often will bleed due to weakness of the new vessel walls. This bleeding causes damage to the retina and kidneys with resulting blindness and renal shutdown, the latter results in required dialysis. Cataracts occur with increasing frequency as the GSH deficiency deepens.
Large and medium sized arteries become sites of accelerated, severe atherosclerosis, with myocardial infarcts at early ages, and of a more severe degree. If diabetics go into heart failure, their mortality rates at one year later are far greater than in non-diabetics. Further, if coronary angioplasty is used to treat their severe atherosclerosis, diabetics are much more likely to have renarrowing of cardiac vessels, termed restenosis.
The above complications are due, in large measure, to GSH deficiency and ongoing free radical reactions. These sequellae frequently and eventually occur despite the use of insulin injections daily that lower blood sugar levels. Good control of blood sugar levels is difficult for the majority of diabetics.
3) Macular Degeneration
Approximately 1 million people in the United States have significant macular degeneration. One out of every 4 persons aged 55 or above now has maculir degeneration and 1 in 2 above the age of 80. As our population ages, this principal cause of blindness in the elderly will increase as well. By the year 2002, 15 million people in the U.S. will suffer from macular degeneration.
Age-related macular degeneration (ARMD) is the disease characterized by either a slow (dry form) or rapid (wet form) onset of destruction and irrevocable loss of rods and cones in the macula of the eye. The macula is the approximate center of the retina wherein the lens of the eye focuses its most intense light. The visual cells, known as the rods and cones, are an outgrowth and active part of the central nervous system. They are responsible and essential for the fine visual discrimination required to see clear details such as faces and facial expression, reading, driving, operation of machinery and electrical equipment and general recognition of surroundings. Ultimately, the destruction of the rods and cones leads to functional, legal blindness. Since there is no overt pain associated with the condition, the first warnings of onset are usually noticeable loss of visual acuity. This may already signal late stage events. It is now thought that one of the very first events in this pathologic process is the formation of a material called "drusen".
Drusen first appears as either patches or diffuse drops of yellow material deposited upon the surface of the retina in the macula lutea or yellow spot. This is the area of the retina there sunlight is focused by the lens. It is the area of the retina that contains the highest density of rods for acuity. Although cones, which detect color, are lost as well in this disease, it is believed to be loss of rods that causes the blindness. Drusen has been chemically analyzed and found to be composed of a mixture of lipids, much of which are peroxidized by free radical reactions. The Drusen first appears as small collections of material at the base of Bruch's membrane. This produces "bubbles" which push the first layer of cells up off the membrane. Vascular budding, neovascular growth, first appears in these channels.
This first layer of cells is unique. They are retinal pigmented epithelial (RPE) cells and these cells are distantly related to CNS microglia and have a phagocytic function. They are also the layer of cells immediately below the primary retinal cells, the rods and cones. The RPE cells are believed to serve a protective function for the rods and cones since they consume the debris cast off by the rods and cones. It is not known yet whether the pigmented material serves a protective function or is related to phagocytosis only. However, this pigment, although concentrated in organelles, is believed to be composed of peroxidized lipids and melanin.
It is believed, because of the order of events in model systems, that the loss of RPE cells occurs first in ARMD (Age Related Macular Degeneration). Once an area of the retinal macula is devoid of RPE cells, loss of rods, and eventually some cones, occurs. Finally, budding of capillaries begins and we see the typical microangiopathy associated with late stage ARMD. It is also known that RPE cells require large quantities of GSH for their proper functioning. When GSH levels drop severely in these cells, in cell cultures where they can be studied, these cells begin to die. When cultures of these cells are supplemented with GSH in the medium, they thrive. There is increasing evidence that progression of the disease is paced by a more profound deficiency in GSH within the retina and probably within these cells, as indicated by cell culture studies.
It is generally believed that "near" ultraviolet (UVB) and visual light of high intensity primarily from sunlight is a strong contributing factor of ARMD. People with light-colored irises constitute a population at high risk, as do those with jobs that leave them outdoors and in equatorial areas where sunlight is most intense. Additional free radical insults, like smoking, add to the risk of developing ARMD.
Several approaches have been recently tested, including chemotherapy, without success. Currently, there is no effective therapy to treat ARMD. Laser therapy has been developed which has been used widely to slow the damage produced in the slow onset form of the disease by cauterizing neovascular growth. However the eventual outcome of the disease, once it has started to progress, is certain.
4) Cellular Regulation by Reactive Oxygen Species
There are a number of types of messengers carrying signals between cells. One type of messenger which has received significant attention recently are small molecule oxidative or free radical agents, which include reactive oxygen species (ROS). These messengers often act by a non-specific interaction with biological macromolecules which may result in a change in configuration. For example, protein secondary structure is typically controlled by cysteine residues, which are susceptible to oxidation with the formation of disulfide bonds. Oxidization of these bonds forming linkages may result in substantial changes in protein configuration and function.
It has thus become increasingly apparent that O.sub.2.sup.- and H.sub.2 O.sub.2 are signaling molecules, changing the behavior of proteins as diverse as transcription factors and membrane receptors by virtue of their ability to undergo redox reactions with the proteins with which they interact, converting --SH groups to disulfide bonds, for example, and changing the oxidation states of enzyme-associated transition metals. As signaling molecules, O.sub.2.sup.- and H.sub.2 O.sub.2 are manufactured by several types of cells, including fibroblasts, endothelial and vascular smooth muscle cells, neurons, ova, spermatozoa and cells of the carotid body. All these cell types appear to use an NAD(P)H oxidase similar to the classical leukocyte NADPH oxidase to produce these oxidants. The stimuli that elicit oxidant production, however, and the purposes for which the oxidants are employed, vary from cell to cell.
Fibroblasts manufacture small but significant amounts of O.sub.2.sup.- in response to inflammatory mediators such as N-formylated peptides and interleukin-1. The O.sub.2.sup.- produced by these cells has been postulated to function as a signaling molecule. Optical spectroscopy has shown that fibroblast membranes contain a heme protein that is different from the flavocytochrome subunit of the leukocyte NADPH oxidase but has properties very similar to those of the leukocyte protein. This heme protein has been suggested as the source of the O.sub.2.sup.- made by these cells.
Endothelial and vascular smooth muscle cells use an NAD(P)H oxidase to produce O.sub.2.sup.- in response to angiotensin II, a peptide hormone that increases blood pressure. This increase in blood pressure appears to be due to the consumption by O.sub.2.sup.- of the NO. that is generated on a continuing basis by the endothelial cells. The resulting fall in NO. concentration raises blood pressure by attenuating or eliminating the vasodilatory effect of NO. that normally prevails in the vascular tree.
Neuronal cells in culture produce oxidants when exposed to amyloid .beta.-peptide, found in amyloid deposits seen in the brains of patients with Alzheimer's disease, or related peptides from other amyloid diseases. The possibility that this O.sub.2.sup.- is produced by an NADPH oxidase is suggested by the observation that flavoprotein inhibitors known to act on the leukocyte NADPH oxidase also inhibit oxidant production in this system. The production of oxidants may be part of a defense used by the neuron against the peptide, with these oxidants perhaps reacting with the peptide to render it susceptible to proteolytic cleavage.
At the moment of fertilization, a membrane NADPH oxidase in sea urchin ova is activated to produce large amounts of H.sub.2 O.sub.2. This oxidant cross-links the proteins of the fertilization membrane by forming dityrosyl bridges, making the membrane impermeable to spermatozoa and thereby preventing polyspermy. This mechanism is common to other species
O.sub.2.sup.- appears to be necessary for the normal function of spermatozoa. When stimulated by a calcium ionophore, normal spermatozoa generate a 3- to 5-min burst of O.sub.2. The O.sub.2.sup.- produced in this reaction is involved in capacitation of the spermatozoa, because the acrosomal response to a number of stimuli is suppressed by superoxide dismutase. On the other hand, spermatozoa that produce O.sub.2.sup.- without stimulation are functionally abnormal, perhaps because of a generalized disruption in their signaling machinery.
The carotid body is a small organ located at the bifurcation of the common carotid artery that measures the oxygen tension of the blood. This organ manufactures H.sub.2 O.sub.2 on a continuing basis, and immunological analysis has shown that its cells contain all 4 of the specific subunits of the leukocyte NADPH oxidase, or proteins very closely related to those subunits. It has been postulated that a carotid body NADPH oxidase very similar or identical to the leukocyte NADPH oxidase is a key component of the oxygen-measuring apparatus of the carotid body.
Thus, in addition to phosphorylation as a control mechanism over regulatory protein configuration and function, reactive oxygen species may also play an important role in cellular regulation and signaling. Selective cysteine oxidation-reduction also serves as an important mechanism for post-translational modification of protein function. This mechanism, termed "redox regulation", has been implicated in a variety of cellular processes such as DNA synthesis, enzyme activation, gene expression, and cell cycle regulation.
Thioredoxin (TRX) is a pleiotropic cellular factor which has thiol-mediated redox activity and plays important roles in regulation of cellular processes, including gene expression. TRX exists either in a reduced, or oxidized form and participates in redox reactions through the reversible oxidation of this active center dithiol. Activity of a number of transcription factors is post-translationally altered by redox modification(s) of specific cysteine residue(s). One such factor is NF-.kappa.B, whose DNA-binding activity is altered by TRX treatment in vitro. The DNA-binding activity of AP-1 is modified by a DNA repair enzyme, Redox Factor-1 (Ref-1). Ref-1 activity is in turn modified by various redox-active compounds, including TRX. TRX translocates from the cytoplasm into the nucleus in response to PMA treatment to associate directly with Ref-1 and modulates not only the DNA-binding but also the transcriptional activity of the AP-1 molecule.
Human thioredoxin (hTRX) has thus been shown to be an important redox regulator in those biological processes. hTRX can function directly by interacting with the target molecules such as NF-.kappa.B transcription factor, or indirectly via another redox protein known as redox factor 1 (Ref-1). Structural Basis Of Thioredoxin-Mediated Redox-Regulation, Qin et al, (poster), Proceedings of 3rd Internet World Congress on Biomedical Sciences, 1996, 12, 9-20 Riken, Tsukuba, Japan. See, also:
Abate, C., Patel, L., Rauscher III, R. J., and Curran, T. (1990) Redox regulation of Fos and Jun DNA binding activity in vitro. Science 249, 1157-1161.
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Cellular redox status modulates various aspects of cellular events including proliferation and apoptosis. TRX is a small (13 kDa), ubiquitous protein with two redox-active half-cystine residues in an active center, -Trp-Cys-Gly-Pro-Cys-, and is also known as adult T-cell leukemia-derived factor (ADF) involved in HTLV-I leukemogenesis. The pathway for the reduction of a protein disulfide by TRX entails nucleophilic attack by one of the active-site sulfhydryls to form a protein-protein disulfide followed by intramolecular displacement of the reduced target proteins with concomitant formation of oxidized TRX. Besides the activity as an autocrine growth factor for HTLV-I-infected T cells and Epstein-Barr virus-transformed lymphocytes, numerous studies have shown the importance of ADF/TRX as a cellular reducing catalyst in human physiology.
In vitro and in vivo experiments showed that TRX augmented the DNA-binding and transcriptional activities of the p50 subunit of NF-.kappa.B by reducing Cys 62 of p50. Direct physical association of TRX and an oligopeptide from NF-.kappa.B p50 has been revealed by NMR study in vitro. Redox regulation of Jun and Fos molecules has also been implicated. Various antioxidants strongly activate the DNA-binding and transactivation abilities of AP-1 complex. TRX enhances the DNA-binding activity of Jun and Fos, in a process which requires other molecules, such as redox factor-1 (Ref-1).
NF-.kappa.B regulates expression of a wide variety of cellular and viral genes. These genes include cytokines such as IL-2, IL-6, IL-8, GM-CSF and TNF, cell adhesion molecules such as ICAM-1 and E-selectin, inducible nitric oxidase synthase (iNOS) and viruses such as human immunodeficiency virus (HIV) and cytomegalovirus. Through the causal relationship with these genes, NF-.kappa.B is considered to be causally involved in the currently intractable diseases such as acquired immunodeficiency syndrome (AIDS), hematogenic cancer cell metastasis and rheumatoid arthritis (RA). Although the genes induced by NF-.kappa.B are variable according to the context of cell lineage and are also under the control of the other transcription factors. NF-.kappa.B plays a major role in regulation of these genes and thus contributes a great deal to the pathogenesis. Therefore, biochemical intervention of NF-.kappa.B should conceivably interfere the pathogenic process and would be effective for the treatment.
NF-.kappa.B consists of two subunit molecules, p65 and p50, and usually exists as a molecular complex with an inhibitory molecule, I.kappa.B, in the cytosol. Upon stimulation of the cells such as by proinflammatory cytokines, IL-1 and TNF, I.kappa.B is dissociated and NF-.kappa.B is translocated to the nucleus and activates expression of target genes. Thus activity of NF-.kappa.B itself is regulated by the upstream regulatory mechanism. Not much is know about the upstream signaling cascade. However, there are at least two independent steps in the NF-.kappa.B activation cascade: kinase pathways and redox-signaling pathway. These two distinct pathways are involved in the NF-.kappa.B activation cascade in a coordinate fashion, which may contribute to a fine tune, as well as fail-safe, regulation of NF-.kappa.B activity.
At least two distinct types of kinase pathways are known to be involved in NF-.kappa.B activation: NF-.kappa.B kinase and I.kappa.B kinase. NF-.kappa.B kinase is a 43 kD serine kinase, associated with NF-.kappa.B. This kinase phosphorylates both subunits of NF-.kappa.B and dissociates it from I.kappa.B. There is another kinase or kinases that is known to phosphorylate I.kappa.B. Consistent with these findings, NF-.kappa.B was shown to be phosphorylated in some cell lines and I.kappa.B was phosphorylated in others in response to stimulation with TNF or IL-1. In most of the cases, NF-.kappa.B dissociation by kinase cascade is a primary step of NF-.kappa.B activation.
After dissociation from I.kappa.B, however, NF-.kappa.B must go through the redox regulation by cellular reducing catalyst, thioredoxin (TRX). TRX is known to participate in redox reactions through reversible oxidation of its active center dithiol to a disulfide. Human TRX has been initially identified as a factor responsible for induction of the a subunit of interleukin-2 receptor which is now known to be under the control of NF-.kappa.B. It is known that NF-.kappa.B can not bind to the .kappa.B DNA sequence of the target genes until it is reduced.
NF-.kappa.B appears to have a novel DNA-binding structure called beta-barrel, a group of beta sheets stretching toward the target DNA. There is a loop in the tip of the beta barrel structure that intercalates with the nucleotide bases and is considered to make a direct contact with the DNA. This DNA-binding loop contains the cystein 62 residue of NF-.kappa.B that is likely the target of redox regulation as a proton donor from TRX. A boot-shaped hollow on the surface of TRX containing the redox-active cysteines could stably recognize the DNA-binding loop of p50 and is likely to reduce the oxidized cysteine by donating protons in a structure-dependent way. Therefore, the reduction of NF-.kappa.B by TRX is considered to be specific.
Not much is known about the initiation of the NF-.kappa.B signaling cascades. However, pretreatment of cells with antioxidants such as N-acetyl-cysteine (NAC) or a-lipoic acid blocks NF-.kappa.B. NAC can also block the induction of TRX. Therefore, anti-NF-.kappa.B actions of antioxidants are considered to be two-fold: 1) blocking the signaling immediately downstream of the signal elicitation, and 2) suppression of induction of the redox effector TRX. It is noted that, in mammals without chroic deseases, such as HIV infection, diabetes, etc. which might impair physiologic glutathione metabolism, a strategy for the pharmaceutical administration of other antioxidants which improve glutathione metabolism or compounds which are themselves appropriate antioxidants may be employed. It is noted that NAC has been shown to have certain neurological toxicity in chronic administration, and therefore this compound is likely inappropriate. On the other hand, lipoic acid may be an advantageous antioxidant alone, or in combination with glutathione. Because of the sensitivity of glutathione oral administration to the particular method of administration, alpha-lipoic acid may have to be administered separately.
The intracellular redox cascade involves successive reduction of oxygen by addition of four electrons and redox regulation of a target protein. Among these ROI hydrogen peroxide has a longest half-life and is considered to be a mediator of oxidative signal. On the other hand, cellular reducing system such as TRX counteracts the action of hydrogen peroxide. The intensity of the oxidative signal may be modulated by the internal GSH level. Similarly, total GSH/GSSG content may influence the responsiveness of the cellular redox signaling. Therefore, intracellular cycteine required to produce GSH.
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Membrane receptors and transporters, including, for example, the insulin receptor and receptors for certain neurotransmitters, are regulated by the redox state of the cell. A very large number of enzymes are also regulated by the cell's redox state. A partial list of proteins whose function is regulated by oxidation-reduction is presented in Table 1.
TABLE 1 Some proteins whose function is regulated by the redox state of the cell. References are given within parentheses Enzymes Collagenase (146,147) p21Ras guanine nucleotide-binding protein (148) Protein tyrosine phosphatase (149) p56Lck protein tyrosine kinase (150) Glycogen phosphorylase phosphatase (151) Glycogen synthase (151) Phosphofructokinase (151) Fructose-1,6-bisphosphatase (151) Hexokinase (151) Pyruvate kinase (151,152) Glucose-6-phosphate dehydrogenase (151) 3-Hydroxy-3-methylglutaryl CoA reductase (151) Serotonin N-acetyltransferase (151) Guanylate cyclase (151) Medium-chain fatty acyl CoA dehydrogenase (153) Xanthine dehydrogenase (154) Chloroplast NADP-linked glyceraldehyde-3-phosphate dehydrogenase (155) Chloroplast NADP-linked malate dehydrogenase (155) Chloroplast sedoheptulose bisphosphatase (155) Fructose bisphosphatase (155) NADP-malic enzyme (156) 3.alpha.-Hydroxysteroid dehydrogenase (157) DsbA protein disulfide isomerase from E. coli (158) Creatine kinase (152) Sarcoplasmic reticulum Ca.sup.2+ -ATPase (152) Transcription factors NF-kappa B (128-130) AP-1 (jun/fos) (131) SoxR (132,133) SoxS (134) OxyR (135) Hypoxia-inducible factor 1 (159) Thyroid transcription factor I (160) Glucocorticoid receptor (161) Sp1 (161,162) Receptors NMDA receptor (163) Insulin receptor NMDA receptor (164,165) Ryanodine receptor (166) HoxB5 (167) c-Myb (167,168) v-Rel (167) p53 (169) Isl-1 (170) Others Erythropoietin RNA-binding protein (171)
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Reactive oxygen species (ROS) are implicated in the pathogenesis of a wide variety of human diseases. Recent evidence suggests that at moderately high concentrations, certain forms of ROS such as H.sub.2 O.sub.2 may act as signal transduction messengers. At least two well-defined transcription factors, nuclear factor (NF-.kappa.B) and activator protein (AP)-1 have been identified to be regulated by the intracellular redox state. R. Schreck, P. Rieber & P. A. Baeuerle, Reactive oxygen intermediates as apparently widely used messengers in the activation of the NF-kappa B transcription factor and HIV-1. EMBO J 10: 2247-2258 (1991). Binding sires of the redox-regulated transcription factors NF-.kappa.B and AP-1 are located in the promoter region of a large variety of genes that are directly involved in the pathogenesis of diseases, e.g.. AIDS, cancer, atherosclerosis and diabetic complications. Biochemical and clinical studies have indicated that antioxidant therapy may be useful in the treatment of disease. Critical steps in the signal transduction cascade are sensitive to oxidants and antioxidants. Many basic events of cell regulation such as protein phosphorylation and binding of transcription factors to consensus sites on DNA are driven by physiological oxidant-antioxidant homeostasis, especially by the thiol-disulfide balance. Endogenous glutathione and thioredoxin systems may therefore be considered to be effective regulators of redox-sensitive gene expression. By controlling redox cascades by using antioxidants, for example, treatments for several diseases may be possible, such as hemotogenic cancer cell metastasis and AIDS. Sen, C. K., Packer, L. Antioxidant and redox regulation of gene transcription. FASEB J. 10, 709-720 (1996). See, also:
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The heat-shock (HS) response is a ubiquitous cellular response to stress, involving the transcriptional activation of HS genes. H.sub.2 O.sub.2 has been shown to induce a concentration-dependent transactivation and DNA-binding activity of heat-shock factor-1 (HSF-1). DNA-binding activity was, however, lower with H.sub.2 O.sub.2 than with HS, thus providing evidence of a dual regulation of HSF by oxidants. The effects of H.sub.2 O.sub.2 in vitro were reversed by the sulphydryl reducing agent dithiothreitol and the endogenous reductor thioredoxin (TRX). In addition, TRX also restored the DNA-binding activity of HSF oxidized in vivo, while it was found to be itself induced in vivo by both HS and H.sub.2 O.sub.2. Thus, H.sub.2 O.sub.2 exerts dual effects on the activation and the DNA-binding activity of HSF: on the one hand, H.sub.2 O.sub.2 favours the nuclear translocation of HSF, while on the other, it alters HSF-DNA-binding activity, most likely by oxidizing critical cysteine residues within the DNA-binding domain. HSF thus belongs to the group of ROS-modulated transcription factors. Muriel R. Jacquier-Sarlin and Barbara S. Polla, "Dual regulation of heat-shock transcription factor (HSF) activation and DNA-binding activity by H.sub.2 O.sub.2 : role of thioredoxin". (1996)
The mammalian stress response evokes a series of neuroendocrine responses that activate the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system. Coordinated interactions between stress response systems, occurring at multiple levels including the brain, pituitary gland, adrenal gland, and peripheral tissues, are required for the maintenance of homeostatic plateau. Glucocorticoids, as a major peripheral effector of the HPA axis, play an essential role in re-establishing homeostatic status in every peripheral tissue in human. On the other hand, the adaptive responses are also operated against various intrinsic or extrinsic forces that disturb cellular homeostasis as a part of local host-defense mechanisms at a cellular level. Currently, reduction/oxidation (redox) reactions are intimately involved in the control of biological processes including modulation of the function of transcription factors, e.g., AP-1 and NF-.kappa.B. Cells contain endogenous buffering systems against excessive production of reactive oxygen intermediates (ROIs) to preserve cellular metabolism through the expression and regulation of many enzymes.
Glucocorticoids, on binding to the glucocorticoid receptor (GR), promote the dissociation of heat shock proteins (HSPs), and the ligand-receptor complex translocates to the nucleus then binds to palindromic DNA sequences, called glucocorticoid response elements (GREs). After binding to DNA, the GR differentially regulates target gene expression to produce hormone action, interacting with or without other transcription factors and coactivators/corepressors. The GR has a modular structure mainly consisting of a central DNA binding domain (DBD), nuclear localization signals, a ligand binding domain (LBD), and several transcription activation functions. The human GR contains 20 cysteine residues, concentrated in the central region spanning the DBD and LBD. The cysteine residues in each domain have been shown to be crucial for maintaining both structure and function of those domains. For examples, it has already been shown that conversion of sulfhydryls in the DBD to disulfides blocks GR binding to DNA cellulose, and that metal ions which have high affinity for thiols interfere with the DBD-DNA interaction.
The TRX system operates as an endogenous defense machinery for glucocorticoid-mediated stress responses against oxidative stress. TRX is considered to be involved in transcriptional processes: for example, NF-.kappa.B activation is inhibited, whereas AP-1 activity is induced by TRX. Moreover, the GR in the isolated rat cytosol is shown to be stabilized and maintained in their reduced, ligand-binding form by TRX. The functional interaction between cellular oxistress, TRX, and GR, and indicate that cellular redox state and TRX levels are important determinants of cellular sensitivity to glucocorticoids. Thus, TRX systems may control homeostasis not only by, for example, sequestrating ROIs, but also by fine tuning of hormonal signals. These phenomena appear to be rationale, for example during inflammation, where cells are believed to be exposed to severe oxidative stress, where suppression of glucocorticoid action may potentiate endogenous defense mechanisms and prevent premature termination of the cascade of inflammatory reactions for self defense. Increase in cellular TRX levels may restore the receptor activity and permit the GR to efficiently communicate with target genes. Resultant activation of anti-inflammatory genes and/or repression of inflammatory genes may prevent overshoot of inflammation. This process may be modulated by an alteration of the redox potential of the cell and the concentration of reduced GSH in the intracellular fluid. Yuichi Makino, Kensaku Okamoto, Kiichi Hirota, Junji Yodoi, Kazuhiko Umesono, Isao Makino, and Hirotoshi Tanaka, Cross-Talk between Endocrine Control of Stress Response and Cellular Antioxidant Defense System, Thioredoxin is a Redox-Regulating Cellular Cofactor for Glucocorticoid Hormone Action (Poster), Proceedings of 3rd Internet World Congress on Biomedical Sciences, 1996.12.9-20 Riken, Tsukuba, Japan. Therefore, glucocorticoid function may be modulated by glutathione administration. Thus, treatment of chronic inflammatory conditions, such as rheumatoid arthritis, as well as other immune and autoimmune disorders, may also benefit from treatment with glutathione. See:
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The role of NF-.kappa.B in HIV life cycle is critical especially in virus reactivation process within the latently infected cells has been widely accepted. After activation through intracellular signaling pathways such as those elicited by T cell receptor antigen complex or by receptors for IL-1 or TNF, NF-.kappa.B initiates HIV gene expression by binding to the target DNA element within the promoter region of HIV LTR. Then, the virus-encoded trans-activator Tat is produced and triggers explosive viral replication. Since activation pathway of HIV gene expression by cellular transcription factor NF-.kappa.B conceptually precedes activation by viral trans-activators, it is conceptual to ascribe NF-.kappa.B as a determinant of the maintenance and breakdown of the viral latency. Antioxidants may be effective in treating AIDS by blocking HIV replication.
Another situation where NF-.kappa.B plays a role is hematogenic cancer cell metastasis. NF-.kappa.B induces E-selectin (also known as ELAM-1) on the surface of vascular endothelial cells. Since some cancer cells constitutively express a ligand for E-selectin, called sialyl-LewisX antigen, on their cell surface, induction of E-selectin is considered to be a rate determining step of cancer cell-endothelial cell interaction. For example, when primary human umbilical venous endothelial cells (HUVEC) are treated with IL-1 or TNF, nuclear translocation of NF-.kappa.B is observed, followed by the augmented expression of E-selectin. In one study, the cell-to-cell interaction between HUVEC and QG90 cell, a tumor cell line derived from human small cell carcinoma of the lung expressing sialyl-LewisX antigen was studied, and it was found that IL-1 was able to induce the attachment of cancer cells to HUVEC. However, pretreatment of HUVEC with N-acetylcysteine, aspirin or pentoxyphillin efficiently blocked the cell-to-cell attachment in a dose-dependent manner. Okamoto, T. et al., Oxygen Radicals, Redox Regulation of the NF-kB Signaling and Disease Control by Antioxidants (poster), Proceedings of 3rd Internet World Congress on Biomedical Sciences, 1996.12.9-20 Riken, Tsukuba, Japan. See, also:
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Feuillard J, Gouy H, Bismuth G, Lee L M, Debre P, Korner M. Nf-kappa B activation by tumor necrosis factor alpha in the Jurkat T cell line is independent of protein kinase A, protein kinase C, and Ca (2)-regulated kinase. Cytokine 1991; 3: 257-265.
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Naumann M, Scheidereit C. Activation of NF-kappa B in vivo is regulated by mutiple phosphorylations. EMBO J 1994; 13: 4597-4607.
Li C-C H, Dai R-M, Chen E, Longo D L. Phosphorylation of NF-KB1-p50 is involved in NF-kappa B activation and stable DNA binding. J Biol Chem 1994; 269: 30089-30092. 38. Okamoto T, Ogiwara H, Hayashi T, Mitsui A, Kawabe T, Yodoi J. Human thioredoxin/adult T cell leukemia-derived factor activates the enhancer binding protein of human immunodeficiency virus type 1 bt thiol redox control mechanism. Int Immunol 1992; 4: 811-819.
Hayashi T, Ueno Y, Okamoto T. Oxidoreductive regulation of nuclear factor kappa B. Involvement of a cellular reducing catalyst thioredoxin. J Biol Chem 1993B; 268: 11380-11388.
Tagaya Y, Maeda Y, Mitsui A, Kondo N, Matsui H, Hamuro J, Brown J, Arai K I, Yokota T, Wakasugi H, Yodoi J. ATL-derived factor (ADF), an IL-2 receptor/Tac inducer homologous to thioredoxin: possible involvement of dithiol-reduction in the IL-2 receptor induction. EMBO J 1989; 8: 757-764.
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Pigmented Epithelium Derived Factor
It is well known that solid tumors, such as carcinomas, require neovascularization to continue growth beyond a few millimeters in size. This is because, as with all tissues, they need oxygen and must rid themselves of toxic metabolic products. Further, rapidly growing tumors may have demands well in excess of that of normal tissues due to a high rate of cell replication. Therefore, one technique which has been sought to be employed in fighting tumors is the use of pharmaceuticals and agents that block neovascularization, for example tumor necrosis factor, endostatin, angiostatin, and other agents. One agent that has aroused interest is Pigmented Epithelium Derived Factor (PEDF), a protein of the serine protease inhibitor (serpin) supergene family, but with characteristics of a substrate rather than inhibitor. PEDF was named for its association with the pigmented RPE cells of the macula, described above. See:
Tombran-Tink et al., "Neuronal Differentiation of Retinoblastoma Cells Induced by Medium Conditioned by Human RPE Cells," Investigative Ophthalmology & Visual Science, 30 (8), 1700-1707 (1989);
G Chader, S P Becerra, L Johnson, J Tombran-Tink, F Steele and I Rodriguez, PCT/US95/07201 filed Jun. 6, 1995, published under WO 95/33480 on Dec. 14, 1995;
U.S. Ser. No. 07/952,796 entitled A DNA Clones for the Expression of Pigment Epithelium Derived Growth Factor and Related Proteins, filed Sep. 24, 1992 by Fintan R. Steele, Gerald J. Chader, Joyce Tombran-Tink and Sofia P. Becerra;
U.S. Ser. No. 08/257,963 entitled A Pigment Epithelium Derived Factor: Characterizations of Its Biological Activity and Sequences Encoding and Expressing the Protein, filed Jun. 7, 1994 by Gerald J. Chader, Sofia P. Becerra, Joan P. Schwartz, Takayuki Taniwaki and Yukihera Sugita, now U.S. Pat. No. 5,840,686;
U.S. Ser. No. 08/279,979 entitled A Retinal Pigmented Epithelium Derived Neurotrophic Factor, filed Jul. 25, 1994 by Fintan R. Steele, Gerald J. Chader, Joyce Tombran-Tink, Sofia P. Becerra and Ignacio R. Rodriquez and Lincoln Johnson;
U.S. Ser. No. 08/367,841 entitled A Pigment Epithelium Derived Factor: Characterization, Genomic Organization and Sequence of the PEDF Gene, filed Dec. 30, 1994 by Gerald J. Chader, Joyce Tombran-Tink, Sofia P. Becerra, Ignacio R. Rodriquez and Fintan R. Steele and Lincoln Johnson;
U.S. Ser. No. 08/377,710 entitled A DNA Clones for the Expression of Pigment Epithelium Derived Factor and Related Proteins, filed Jan. 25, 1995 by Fintan R. Steele, Gerald J. Chader, Joyce Tombran-Tink, Sofia P. Becerra and Ignacio R. Rodriquez;
U.S. Ser. No. 08/520,373 entitled A Retinal Pigmented Epithelium Derived Neurotrophic Factor, filed Aug. 29, 1995 by Gerald J. Chader, Joyce Tombran-Tink, Sofia P. Becerra, Ignacio R. Rodriquez and Fintan R. Steele;
F R Steele, G J Chader, L V Johnson, J Tombran-Tink. Pigment epithelium-derived factor: neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1526-1530;
S P Becerra, I Palmer, A Kumar, F Steele, J Shiloach, V Notario, G J Chader. Overexpression of fetal human pigment epithelium-derived factor in Escherichia coil: a functionally active neurotrophic factor. J Biol Chem Nov. 5, 1993; 268 (31): 23148-56;
Perez-Mediavilla L A, Chew C, Campochiaro P A, Nickells R W, Notario V, Zack D J, Becerra S P. Sequence and expression analysis of bovine pigment epithelium-derived factor. Biochim Biophys Acta Jun. 16, 1998; 1398 (2): 203-14;
Slavc I; Rodriguez I R; Mazuruk K; Chader G J; Biegel J A. Mutation analysis and loss of heterozygosity of PEDF in central nervous system primitive neuroectodermal tumors, Int J Cancer 1997; 72 (2): 277-82.
PEDF is a potent autocrine and paracrine hormone which blocks epithelial cell proliferation (including vascular epithelial cells, necessary for neovascularization), and promotes cellular differentiation, and is neurotrophic and neuroprotective. Sugita Y, Becerra S P, Chader G J, Schwartz J P, Pigment epithelium-derived factor (PEDF) has direct effects on the metabolism and proliferation of microglia and indirect effects on astrocytes., J Neurosci Res Sep. 15, 1997; 49 (6): 710-8. Subsequent studies have confirmed that PEDF or its isoforms are widely distributed throughout the body, but with relatively high concentration in the pigmented epithelial cells of the retina and central nervous system. PEDF may help cells resist apoptosis. Araki T, Taniwaki T, Becerra S P, Chader G J, Schwartz J P, Pigment epithelium-derived factor (PEDF) differentially protects immature but not mature cerebellar granule cells against apoptotic cell death, J Neurosci Res Jul. 1, 1998; 53 (1): 7-15. Glutathione depletion has also been directly associated with failure of differentiation. Esposito F, Agosti V, Morrone G, Morra F, Cuomo C, Russo T, Venuta S, Cimino F, Inhibition of the differentiation of human myeloid cell lines by redox changes induced through glutathione depletion, Biochem. J. (1994) 301, 649-653.
PEDF binds to extracellular matrixes. Alberdi E, Hyde C C, Becerra S P, Pigment epithelium-derived factor (PEDF) binds to glycosaminoglycans: analysis of the binding site. Biochemistry Jul. 28, 1998; 37 (30): 10643-52 (Published erratum appears in Biochemistry Dec. 22, 1998; 37 (51): 18128).
PEDF is among the most potent direct angiogenesis factors known. In the eye, it prevents ingrowth of blood vessels in the lens, retina and vitreous body of the eye. Ortego J, Escribano J, Becerra S P, Coca-Prados M, Gene expression of the neurotrophic pigment epithelium-derived factor in the human ciliary epithelium. Synthesis and secretion into the aqueous humor, Invest Ophthalmol Vis Sci December 1996; 37 (13): 2759-67. PEDF is a dramatic enhancer of cellular differentiation, and is capable, for example, of inducing retinoblastoma cells to retrotransform into normal appearing cells. Stratikos E, Alberdi E, Gettins P G, Becerra S P, Recombinant human pigment epithelium-derived factor (PEDF): characterization of PEDF overexpressed and secreted by eukaryotic cells. Protein Sci December 1996; 5 (12): 2575-82. PEDF protects neural tissue against an array of injurious factors, for example, against the excitatory neurotoxicity of glutamate. Taniwaki T, Hirashima N, Becerra S P, Chader G J, Etcheberrigaray R, Schwartz J P. Pigment epithelium-derived factor protects cultured cerebellar granule cells against glutamate-induced neurotoxicity. J Neurochem January 1997; 68 (1): 26-32.
PEDF is produced by the stromal cells of the endometrium and has a strong effect on the growth and differentiation of the glandular epithelium. When stromal cells become deciduous cells, in response to hormones and pregnancy, PEDF production is considered crucial to prevent (i) uncontrolled growth and penetration of the otherwise highly invasive trophoblastic cells of the placenta, into the uterine wall, and (ii) uncontrolled ingrowth of the blood vessels from the chorionic villi, into the uterine wall.
PEDF controls the cell cycle in many different cell types, by a direct effect on cell cycle control factors. The source of PEDF, namely the retinal pigment epithelium (RPE), may be crucial to the normal development and function of the neural retina. A variety of biologically active molecules, including growth factors, are synthesized and secreted by RPE cells. The RPE develops prior to and lies adjacent to the neural retina, and that it functions as part of the blood-retina barrier. Fine et al., The Retina, Ocular Histology: A Text and Atlas, New York, Harper & Row, 61-70 (1979), the RPE has been implicated in vascular, inflammatory, degenerative, and dystrophic diseases of the eye. Elner et al., Am. J. Pathol., 136, 745-750 (1990). In addition to growth factors, nutrients and metabolites are also exchanged between the RPE and the retina. For example, the RPE supplies to the retina the well-known growth factors PDGF, FGF, TGF.alpha., and TGF.beta.. Campochiaro et al., Invest. Ophthalmol. Vis. Sci., 29, 305-311 (1988): Plouet, Invest. Ophthalmol. Vis. Sci., 29, 106-114 (1988); Fassio et al., Invest. Ophthalmol. Vis. Sci., 29, 242-250 (1988); Connor et al., Invest. Ophthalmol. Vis. Sci., 29, 307-313 (1988). It is very likely that these and other unknown factors supplied by the RPE influence the organization, differentiation, and normal functioning of the retina.
In order to study and determine the effects of putative differentiation factors secreted by the RPE, cultured cells have been subjected to retinal extracts and conditioned medium obtained from cultures of human fetal RPE cells. For example, U.S. Pat. No. 4,996,159 (Glaser) discloses a neovascularization inhibitor recovered from RPE cells that is of a molecular weight of about 57,000.+-.3,000. Similarly, U.S. Pat. Nos. 1,700,691 (Stuart), 4,477,435 (Courtois et al.), and 4,670,257 (Guedon born Saglier et al.) disclose retinal extracts and the use of these extracts for cellular regeneration and treatment of ocular disease. Furthermore, U.S. Pat. Nos. 4,770,877 (Jacobson) and 4,534,967 (Jacobson et al.) describe cell proliferation inhibitors purified from the posterior portion of bovine vitreous humor.
PEDF has been isolated from human RPE as a 50-kDa protein. Tombran-Tink et al., Invest. Ophthalmol. Vis. Sci., 29, 414 (1989); Tombran-Tink et al., Invest. Ohthalmol. Vis. Sci., 30, 1700-1707 (1989); Tombran-Tink et al., "PEDF: A Pigment Epithelium-derived Factor with Potent Neuronal Differentiative Activity," Experimental Eye Research, 53, 411-414 (1991). Specifically, PEDF has been demonstrated to induce the differentiation of human Y79 retinoblastoma cells, which are a neoplastic counterpart of normal retinoblasts. Chader, Cell Different., 20, 209-216 (1987); Taniwaki T, Becerra S P, Chader G J, Schwartz J P, Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture, J Neurochem June 1995; 64 (6): 2509-17. The differentiative changes induced by PEDF include the extension of a complex meshwork of neurites, and expression of neuronal markers such as neuron-specific enolase and neurofilament proteins. This is why the synthesis and secretion of PEDF protein by the RPE is believed to influence the development and differentiation of the neural retina. Furthermore, PEDF is only highly expressed in undifferentiated human retinal cells, like Y79 retinoblastoma cells, but is either absent or downregulated in their differentiated counterparts. It was also reported that PEDF mRNA is expressed in abundance in quiescent human fetal W1 fibroblast cells and not expressed in their senescent counterparts. Pignolo et al. (1993), J. Biol. Chem., 268: 2949-295.
Further study of PEDF and examination of its potential therapeutic use in the treatment of inflammatory, vascular, degenerative, and dystrophic diseases of the retina and central nervous system (CNS) necessitates the obtention of large quantities of PEDF. Unfortunately, the low abundance of PEDF in fetal human eye and, furthermore, the rare availability of its source tissue, especially in light of restrictions on the use of fetal tissue in research and therapeutic applications, make further study of PEDF difficult at best. Therefore, a recombinant technique was developed to procure a supply of the factor. See, U.S. Pat. No. 5,840,686, supra.
Based upon the protein amino acid sequence, PEDF has been found to have extensive sequence homology with the serpin gene family, members of which are serine protease inhibitors. Many members of this family have a strictly conserved domain at the carboxyl terminus which serves as the reactive site of the protein. These proteins are thus thought to be derived from a common ancestral gene. However the developmental regulation differs greatly among members of the serpin gene family and many have deviated from the classical protease inhibitory activity. Becerra S P, Structure-function studies on PEDF. A noninhibitory serpin with neurotrophiic activity, Adv Exp Med Biol 1997; 425: 223-37. Although PEDF shares sequence homology with serpins, analysis of the cDNA sequence indicates that it lacks the conserved domain and thus may not function as a classical prolease inhibitor.
Genomic sequencing and analysis of PEDF has provided sequences of introns and exons as well as approx. 4 kb of 5'-upstream sequence. The gene for PEDF has been localized to 17p13.1 using both in situ hybridization and analyses of somatic cell hybrid panels. Tombran-Tink, et al., (1994) Genomics, 19: 266-272. This is very close to the p53 tumor suppressor gene as well as to the chromosomal localization of a number of hereditary cancers unrelated to mutations in the p53 gene product PEDF thus becomes a prime candidate gene for these cancers.
Although PEDF is particularly highly expressed by RPE cells, it is detectable in most tissues, cell types, tumors, etc. by Northern and Western blot analyses. It is readily detected, for example in vitreous and aqueous humors. The important question of subcellular localization of PEDF has also been addressed. Although the bulk of the PEDF appears to be secreted, PEDF is also associated with the nucleus as well as with very specific cytoskeletal structures in the cytoplasm. Importantly, this varies as to the age of the cells and the specific cell-cycle state. For example, the protein appears to concentrate at the tips of the pseudopods of primate RPE cells that interact with the substratum during the initial stages of attachment. Later though, this staining disappears and there is appearance of the protein in association with specific cytoskeletal structures and the nucleus. Thus it appears that PEDF plays an important intracellular role in both nucleus and cytoplasm.
There is PEDF expression in dividing, undifferentiated Y-79 cells and little or no expression in their quiescent, differentiated counterparts. Tombran-Tink, et al., (1994) Genomics, 19: 266-272. The synthesis of PEDF in WI-38 fibroblast cells is restricted to the G.sub.0 stage of the cell cycle in young cells. Pignolo et al. (1993), J. Biol. Chem., 268: 2949-295. Moreover, in old senescent cells, PEDF messenger RNA is absent.
In the retina, PEDF inhibits the Muller glial cells. Since Muller cells are similar to astroglia, PEDF would be similarly effective in blocking gliosis in conditions such as retinal etachment, diabetes, Retinitis Pigmentosa, etc. as well as sparing the lives of the retinal neurons. Thus, administration of glutathione, to alter cellular redox potential, and thereby alter PEDF expression, may have particular value.
Apparently, in macular degeneration, the pigmented RPE cells become defective, and die, resulting in a functional loss of PEDF in the macula. Without the continuous presence of PEDF, vascular epithelial cells undergo a de-differentiation and enter into a proliferative stage, resulting in neovascularization, with invasion of the cornea in vitreous with blood vessels. The amount of inhibitory PEDF produced by retinal cells is positively correlated with oxygen concentration. Thus, PEDF is presumed to play a role in ischemia-driven retinal neovascularization. In fact, studies have shown that it is not necessary to kill the RPE cells to reduce PEDF availability. The availability of PEDF is sensitive to the redox potential of the cell, being more available in a reduced state and less available when the cell is in an oxidized state. (Ischemia is associated with a state in which cells produce an excess of free radicals. These may be due to exhaustion of antioxidants, cell death or apoptosis, or accumulation of toxic metabolic waste). This feedback regulation, which is applicable to other PEDF producing cells, thus induces vascularization where blood flow is needed (relatively oxidized redox potential) while maintaining an appropriate balance and allowing certain privileged tissues to remain unvascularized or with highly controlled vascularization. The oxidative control over PEDF is believed to be at the translative or post-translative levels, as mRNA levels are generally unchanged. It is noted that other classes of biologically active agents respond to redox state through transcriptional modification or sensitivity.
Efforts to directly administer PEDF, a peptide hormone, are met with difficulty, due to both the unavailability of bulk quantities of PEDF and difficulties in administration thereof.
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Metabolism of Glutathione.
The synthesis of GSH is dependent upon the availability of cysteine either supplied directly from the diet or cysteine or indirectly from methionine via the transsulfuration pathway. GSH synthesis and metabolism is governed by the enzymes of the .gamma.-glutamyl cycle. GSH is synthesized intracellularly by the consecutive actions of .gamma.-glutamylcysteinyl synthetase (Reaction 1) and GSH synthetase (Reaction 2). The action of the latter enzyme is feedback inhibited by GSH. The breakdown of GSH (and also of its oxidized form, GSSG) is catalyzed by .gamma.-glutamyl transpeptidase, which catalyzes the transfer of the gamma-glutamyl moiety to acceptors such as sulfhydryl-containing amino acids, certain dipeptides, and GSH itself (Reaction 3). The cellular turnover of GSH is associated with its transport, in the form of GSH, across cell membranes, where the majority of the transpeptidase is found. During this transport, GSH interacts with .gamma.-glutamyl transferase (also known as transpeptidase) to form .gamma.-glutamyl amino acids which are transported into cells. Intracellular .gamma.-glutamyl amino acids are substrates of .gamma.-glutamyl cyclotransferase (Reaction 4) which converts these compounds into the corresponding amino acids and 5-oxo-L-proline. The ATP-dependent conversion of 5-L-oxoproline to L-glutamate is catalyzed by the intracellular enzyme 5-oxo-prolinase (Reaction 5). The cysteinylglycine formed in the transpeptidase reaction is split by dipeptidase (Reaction 6). These six reactions constitute the .gamma.-glutamyl cycle, which accounts for the synthesis and enzymatic degradation of GSH.
Two of the enzymes of the cycle also function in the metabolism of S-substituted GSH derivatives, which may be formed nonenzymatically by reaction of GSH with certain electrophilic compounds or by GSH S-transferases (Reaction 7). The .gamma.-glutamyl moiety of such conjugates is removed by the action of .gamma.-glutamyl transpeptidase (Reaction 3), a reaction facilitated by .gamma.-glutamyl amino acid formation. The resulting S-substituted cysteinylglycines are cleaved by dipeptidase (Reaction 6A) to yield the corresponding S-substituted cysteines, which may undergo N-acetylation (Reaction 8) or an additional transpeptidation reaction to form the corresponding .gamma.-glutamyl derivative (Reaction 3A).
Intracellular GSH is converted to its oxidized, dimeric form (GSSG) by selenium-containing GSH peroxidase, which catalyzes the reduction of H.sub.2 O.sub.2 and other peroxides (Reaction 9). GSH is also converted to GSSG by transhydrogenation (Reaction 10). Reduction of GSSG to GSH is mediated by the widely-distributed enzyme GSSG reductase which uses NADPH (Reaction 11). Extracellular conversion of GSH to GSSG has also been reported; the overall reaction requires O.sub.2 and leads to the formation of H.sub.2 O.sub.2 (Reaction 12). GSSG is also formed by reaction of GSH with free radicals. The glutathione-dependent antioxidant system consists of glutathione plus two enzymes: glutathione peroxidase and glutathione reductase. As this system operates, glutathione cycles between its oxidized (GSSG) and reduced (GSH) forms.
Lipid hydroperoxides, which are formed during the peroxidation of lipids containing unsaturated fatty acids, are reduced, not by the usual glutathione peroxidase, but by a special enzyme designed specifically to handle peroxidized fatty acids in phospholipids. This enzyme, known as phospholipid hydroperoxide glutathione peroxidase is protein that can reduce both H.sub.2 O.sub.2 and lipid hydroperoxides to the corresponding hydroxides (water and a lipid hydroxide, respectively). In contrast to the phospholipid hydroperoxide glutathione peroxidase, ordinary glutathione peroxidase is unable to act on lipid hydroperoxides.
Transport of Glutathione.
The intracellular level of GSH in mammalian cells is in the range of 0.5-10 millimolar, while micromolar concentrations are typically found in blood plasma. Intracellular glutathione is normally over 99% reduced form (GSH). The normal healthy adult human liver synthesizes 8-10 grams of GSH daily. Normally, there is an appreciable flow of GSH from liver into plasma. The major organs involved in the inter-organ transport of GSH are the liver and the kidney, which is the primary organ for clearance of circulating GSH. It has been estimated to account for 50-67% of net plasma GSH turnover. Several investigators have found that during a single pass through the kidney, 80% or more of the plasma GSH is extracted, greatly exceeding the amount which could be accounted for by glomerular filtration. While the filtered GSH is degraded stepwise by the action of the brush-border enzymes .gamma.-glutamyltransferase and cysteinylglycine dipeptidase, the remainder of the GSH appears to be transported via an unrelated, Na+-dependent system present in basal-lateral membranes.
GSH transported from hepatocytes interacts with the transpeptidase of ductile cells, and there appears to be a substantial reabsorption of metabolites by ductule endothelium. In the rat, about 12 and 4 nmoles/g/min of GSH appear in the hepatic vein and bile, respectively.
Glutathione exists in plasma in four forms: reduced glutathione (GSH), oxidized glutathione (GSSG), mixed disulfide with cysteine (CySSG) and protein bound through a sulfhydryl linkage (GSSPr). The distribution of glutathione equivalents is significantly different than that of cyst(e)ine, and when either GSH or cysteine is added at physiological concentration, a rapid redistribution occurs. The distribution of glutathione equivalents in rat plasma is 70.0% protein bound, with the remaining 30% apportioned as follows: 28.0% GSH, 9.5% GSSG, and 62.6% as the mixed disulfide with cysteine. The distribution of cysteine equivalents was found to be 23% protein bound, with the remaining 77% distributed as follows: 5.9% cysteine, 83.1% cystine, and 10.8% as the mixed disulfide with glutathione. Plasma thiols and disulfides are not in equilibrium, but appear to be in a steady state maintained in part by transport of these compounds between tissues during the interorgan phase of their metabolism. The large amounts of protein-bound glutathione and cysteine provide substantial buffering which must be considered in the analysis of transient changes in glutathione and cysteine. This buffering may protect against transient thiol-disulfide redox changes which could affect the structure and activity of plasma and plasma membrane proteins. In erythrocytes, GSH has been implicated in reactions which maintain the native structure of hemoglobin and of enzymes and membrane proteins. GSH is present in erythrocytes at levels 1000 times greater than in plasma. It functions as the major small molecule antioxidant defense against toxic free radicals, an inevitable by-product of the erythrocytes' handling of O.sub.2.
Glutathione and the Immune System.
The importance of thiols and especially of GSH to lymphocyte function has been known for many years. Adequate concentrations of GSH are required for mixed lymphocyte reactions, T-cell proliferation, T- and B- cell differentiation, cytotoxic T-cell activity, and natural killer cell activity. Adequate GSH levels have been shown to be necessary for microtubule polymerization in neutrophils. Intraperitoneally administered GSH augments the activation of cytotoxic T-lymphocytes in mice, and dietary GSH was found to improve the splenic status of GSH in aging mice, and to enhance T-cell-mediated immune responses.
The presence of macrophages can cause a substantial increase of the intracellular GSH levels of activated lymphocytes in their vicinity. Macrophages consume cystine via a strong membrane transport system, and generate large amounts of cysteine which they release into the extracellular space. It has been demonstrated that macrophage GSH levels (and therefore cysteine equivalents) can be augmented by exogenous GSH. T-cells cannot produce their own cysteine, and it is required by T-cells as the rate-limiting precursor of GSH synthesis. The intracellular GSH level and the DNA synthesis activity in mitogenically-stimulated lymphocytes are strongly increased by exogenous cysteine, but not cystine. In T-cells, the membrane transport activity for cystine is ten-fold lower than that for cysteine. As a consequence, T-cells have a low baseline supply of cysteine, even under healthy physiological conditions. The cysteine supply function of the macrophages is an important part of the mechanism which enables T-cells to shift from a GSH-poor to a GSH-rich state.
The importance of the intracellular GSH concentration for the activation of T-cells is well established. It has been reported that GSH levels in T-cells rise after treatment with GSH; it is unclear whether this increase is due to uptake of the intact GSH or via extracellular breakdown, transport of breakdown products, and subsequent intracellular GSH synthesis. Decreasing GSH by 10-40% can completely inhibit T-cell activation in vitro. Depletion of intracellular GSH has been shown to inhibit the mitogenically-induced nuclear size transformation in the early phase of the response. Cysteine and GSH depletion also affects the function of activated T-cells, such as cycling T-cell clones and activated cytotoxic T-lymphocyte precursor cells in the late phase of the allogenic mixed lymphocyte culture. DNA synthesis and protein synthesis in IL-2 dependent T-cell clones, as well as the continued growth of preactivated CTL precursor cells and/or their functional differentiation into cytotoxic effector cells are strongly sensitive to GSH depletion.
The activation of physiologic activity of mouse cytotoxic T-lymphocytes in vivo was found to be augmented by interperitoneal (i.p.) GSH in the late phase but not in the early phase of the response. The injection of GSH on the third day post immunization mediated a 5-fold augmentation of cytotoxic activity. Dietary GSH supplementation can reverse age-associated decline of immune response in rats, as demonstrated by maintenance of Concanavalin A stimulated proliferation of splenocytes in older rats.
Glutathione status is a major determinant of protection against oxidative injury. GSH acts on the one hand by reducing hydrogen peroxide and organic hydroperoxides in reactions catalyzed by glutathione peroxidases, and on the other hand by conjugating with electropililic xenobiotic intermediates capable of inducing oxidant stress. The epithelial cells of the renal tubule have a high concentration of GSH, no doubt because the kidneys function in toxin and waste elimination, and the epithelium of the renal tubule is exposed to a variety of toxic compounds. GSH, transported into cells from the extracellular medium, substantially protects isolated cells from intestine and lung are against t-butylhydroperoxide, menadione or paraquat-induced toxicity. Isolated kidney cells also transport GSH, which can supplement endogenous synthesis of GSH to protect against oxidant injury. Hepatic GSH content has also been reported to rise, indeed to double, in the presence of exogenous GSH. This may be due either to direct transport, as has been reported for intestinal and alveolar cells, or via extracellular degradation, transport, and intracellular resynthesis.
The nucleophilic sulfur atom of the cysteine moiety of GSH serves as a mechanism to protect cells from harmful effects induced by toxic electrophiles. The concept that glutathione S-conjugate biosynthesis is an important mechanism of drug and chemical detoxification is well established. GSH conjugation of a substrate generally requires both GSH and glutathione-S-transferase activity. The existence of multiple glutathione-S-transferases with specific, but also overlapping, substrate specificities enables the enzyme system to handle a wide range of compounds.
Several classes of compounds are believed to be converted by glutathione conjugate formation to toxic metabolites. Halogenated alkenes, hydroquinones, and quinones have been shown to form toxic metabolites via the formation of S-conjugates with GSH. The kidney is the main target organ for compounds metabolized by this pathway. Selective toxicity to the kidney is the result of the kidney's ability to accumulate intermediates formed by the processing of S-conjugates in the proximal tubular cells, and to bioactivate these intermediates to toxic metabolites.
The administration of morphine and related compounds to rats and mice results in a loss of up to approximately 50% of hepatic GSH. Morphine is known to be biotransformed into morphinone, a highly hepatotoxic compound, which is 9 times more toxic than morphine in mouse by subcutaneous injection, by morphine 6-dehydrogenase activity. Morphinone possesses an .alpha.,.beta.-unsaturated ketone, which allows it to form a glutathione S-conjugate. The formation of this conjugate correlates with loss of cellular GSH. This pathway represents the main detoxification process for morphine. Pretreatment with GSH protects against morphine-induced lethality in the mouse.
The deleterious effects of methylmercury on mouse neuroblastoma cells are largely prevented by coadministration of GSH. GSH may complex with methylmercury, prevent its transport into the cell, and increase cellular antioxidant capabilities to prevent cell damage. Methylmercury is believed to exert its deleterious effects on cellular microtubules via oxidation of tubulin sulfhydryls, and by alterations due to peroxidative injury. GSH also protects against poisoning by other heavy metals such as nickel and cadmium.
Because of its known role in renal detoxification and its low toxicity, GSH has been explored as an adjunct therapy for patients undergoing cancer chemotherapy with nephrotoxic agents such as cisplatin, in order to reduce systemic toxicity. In one study, GSH was administered intravenously to patients with advanced neoplastic disease, in two divided doses of 2,500 mg, shortly before and after doses of cyclophosphamide. GSH was well-tolerated and did not produce unexpected toxicity. The lack of bladder damage, including microscopic hematuria, supports the protective role of this compound. Other studies have shown that i.v. GSH coadministration with cisplatin and/or cyclophosphamide combination therapy, reduces associated nephrotoxicity, while not unduly interfering with the desired cytotoxic effect of these drugs.
Bohm, S., Battista-Spatti, G., DiRe, F., Oriana, S., Pilotti, S., Tedeschi, M., Tognella, S. & Zunino, F.: A feasibility study of cisplatin administration with low-volume hydration and glutathione protection in the treatment of ovarian carcinoma. Anticancer Res. 11: 1613-1616. 1991.
Cozzaglio, L., Doci, R., Colla, G., Zunino, F., Casciarri, G. & Gennari, L.: A feasibility study of high-dose cisplatin and 5-fluorouracil with glutathione protection in the treatment of advanced colorectal cancer. Tumori 76: 590-594, 1990.
Di Re, F., Bohm, S., Oriana, S., Spatti, G.B., & Zunino, F.: Efficacy and safety of high-dose cisplatin and cyclophosphamide with glutathione protection in the treatment of bulky advanced epithelial ovarian cancer. Cancer Chemother. Pharmacol. 25: 355-360, 1990.
Nobile, M. T., Vidili, M. G., Benasso, M., Venturini, M., Tedeschi, M., Zunino, F., & Rosso, R.: A preliminary clinical study of cyclophosphamide with reduced glutathione as uroprotector. Tumori 75: 257-258, 1989.
Clinical Use of Glutathione
Ten elderly patients with normal glucose tolerance and ten elderly patients with impaired glucose tolerance (IGT) underwent GSH infusion, 10 mg/min for 120 min, for a total dose of 1,200 mg in 2 hr, under basal conditions and during 75 g oral glucose tolerance tests and intravenous glucose tolerance tests. Basal plasma total glutathione levels were essentially the same for normal and IGT groups, and GSH infusion under basal conditions increased GSH to similar levels. This study demonstrated that GSH significantly potentiated glucose-induced insulin secretion in patients with IGT. No effect was seen on insulin clearance and action.
The antihypertensive effect of an i.v. bolus of 1,844 mg. or 3,688 mg. GSH was studied in normal and mild to moderate essential hypertensive subjects and in both hypertensive and non-hypertensive diabetics, both type I and type II. The administration of 1,844 mg. GSH produced a rapid and significant decrease in both systolic and diastolic blood pressure, within ten minutes, but which returned to baseline within 30 minutes, in both groups of hypertensive patients and in non-hypertensive diabetics, but had no effect in normal healthy subjects. At the 3,699 mg. dose, not only did the blood pressure decrease in the hypertensive subjects, but GSH produced a significant decrease in the blood pressure values in normal subjects as well.
GSH, 1,200 mg/day intravenously administered to chronic renal failure patients on hemodialysis was found to significantly increase studied hematologic parameters (hematocrit, hemoglobin, blood count) as compared to baseline, and holds promise to reverse the anemia seen in these patients.
See, Costagliola, C., Romano, L., Scibelli, G., de Vincentiis, A., Sorice, P. & DiBenidetto, A.: Anemia and chronic renal failure: a therapeutic approach by reduced glutathione parenteral administration. Nephron 61: 404-408, 1992.
Toxicological Effects of Glutathione.
The reported LD.sub.50 of GSH in rats and mice via various routes of administration are listed in the table below. GSH has an extremely low toxicity, and oral LD.sub.50 measurements are difficult to perform due to the sheer mass of GSH which has to be ingested by the animal in order to see any toxic effects.
 Route of Animal Admin. LD.sub.50 Reference Mouse Oral 5000 mg/kg Modern Pharmaceuticals of Japan, IV Edition. Tokyo, Japan Pharmaceutical, Medical and Dental Supply Exporters' Association, 1972, p 93. Mouse Intraperitoneal 4020 mg/kg Modern Pharmaceuticals of Japan, IV Edition. Tokyo, Japan Pharmaceutical. Medical and Dental Supply Exporters' Association. 1972. p 93. Mouse Intraperitoneal 6815 mg/kg Toxicology, vol. 62. p. 205, 1990. Mouse Subcutaneous 5000 mg/kg Modern Pharmaceuticals of Japan, IV Edition. Tokyo, Japan Pharmaceutical. Medical and Dental Supply Exporters' Association. 1972. p 93. Mouse Intravenous 2238 mg/kg Japanese J. of Antibiotics, vol. 38. p. 137. 1985. Mouse Intramuscular 4000 mg/kg Modern Pharmaceuticals of Japan, III Edition. Tokyo, Japan Pharmaceutical. Medical and Dental Supply Exporters' Association. 1968. p 97.
GSH can be toxic, especially in cases of ascorbate deficiency, and these effects may be demonstrated in, for example, ascorbate deficient guinea pigs given 3.75 mmol/kg daily (1,152 mg/kg daily) in three divided doses, whereas in non-ascorbate deficient animals, toxicity was not seen at this dose, but were seen at double this dose. See:
Dalhoff, K., Ranek, L., Mantoni, M. & Poulsen, H. E.: Glutathione treatment of hepatocellular carcinoma. Liver 12: 341-343, 1992.
Dekant, W.: Bioactivation of nephrotoxins and renal carcinogens by glutathione S-conjugate formation. Toxicol. Letters 67: 151-60, 1993.
Domingo, J. L., Gomez, M., Llobet, J. M. & Corbella, J.: Chelating agents in the treatment of acute vanadyl sulphate intoxication in mice. Toxicology 62: 203-211, 1990.
Martensson, J., Han, J., Griffith, O. W. & Meister, A.: Glutathione ester delays the onset of scurvy in ascorbate-deficient guinea pigs. Proc. Nat. Acad. Sci. USA 90: 317-321, 1993.
Thust, R, and Bach, B.: Exogenous glutathione induces sister chromatid exchanges, clastogenicity and endoreduplication in V79-E Chinese hamster cells. Cell Biol. Toxicol. 1: 123-31, 1985.
Aebi, S. & Lauterberg, B. H.: Divergent effects of intravenous GSH and cysteine on renal and hepatic GSH. Aer. J. Physiol. 263 (2 pt 2): R348-R352, 1992.
Ammon, H. P. T., Melien, M. C. M. & Verspohl, E. J.: Pharmacokinetics of intravenously administered glutathione in the rat. J. Pharm. Pharmacol. 38: 721-725, 1986.
Anderson, M. E., Powrie, F., Puri, R. N., & Meister, A.: Glutathione monoethyl ester: Preparation, uptake by tissues, and conversion to glutathione. Arch. Biochem. Biophys. 239: 538-548, 1985.
Aw, T. Y., Wierzbicka, G. & Jones, D. P.: Oral glutathione increases tissue glutathione in vivo. Chem. Biol. Interact. 80: 89-97, 1991.
Borok, Z., Buhl, R., Grimes, G. J., Bokser, A. D., Hubbard, R. C., Holroyd, K. J., Roum, J. H., Czerski, D. B., Cantin, A. M., & Crystal, R. G.: Effect of glutathione aerosol on oxidant-antioxidant imbalance in idiopathic pulmonary fibrosis. The Lancet 338: 215-216, 1991.
Buhl, R., Vogelmeier, C., Critenden, M., Hubbard, R. C., Hoyt, Jr., R. F., Wilson, E. M., Cantin, A. M. & Crystal, R. G.: Augmentation of glutathione in the fluid lining the epithelium of the lower respiratory tract by directly administering glutathione aerosol. Proc. Natl. Acad. Sci. USA 87: 4063-4067, 1990.
Bump, E. A., al-Sarraf, R., Pierce, S. M. & Coleman, C. N.: Elevation of mouse kidney thiol content following administration of glutathione. Radiother. Oncol. 23: 21-25, 1992.
Griffith, O. W., Bridges, R. J., & Meister, A.: Formation of g-glutamyl-cyst(e)ine in vivo is catalyzed by .gamma.-glutamyl transpeptidase. Proc. Natl. Acad. Sci. USA 78: 2777-2781, 1981.
Hagen, T. M., Wierzbicka, G. T., Bowman, B. B., Aw, T. Y. & Jones, D. P.: Fate of dietary glutathione. Disposition in the gastrointestinal tract. Am. J. Physiol. 259: G530-G535, 1990.
Hagen, T. M. & Jones, D. P.: Transepithelial transport of glutathione in vascularly perfused small intestine of rat. Am. J. Physiol. 252: G607-G613, 1987.
Hagen, T. M., Wierzbicka, G. T., Sillau, A. H., Bowman, B. B. & Jones, D. P.: Bioavailability of dietary glutathione. Effect on plasma concentration. Am. J. Physiol. 259: G524-G529, 1990.
Hahn, R., Wendel, A. & Flohe, L.: The fate of extracellular glutathione in the rat. Biochim. Biophys. Acta 539: 324-337, 1978.
Puri, R. N., & Meister, A.: Transport of glutathione, as g-glutamylcysteinylglycyl ester, into liver and kidney. Proc. Natl. Acad. Sci. USA 80: 5258-5260, 1983.
Vina, J., Perez, C., Furukawa, T., Palacin, M. & Vina, J. R.: Effect of oral glutathione on hepatic glutathione levels in rats and mice. Brit. J. Nutr. 62: 683-91, 1989.
Aebi, S., Asserto, R., & Lauterberg, B. H.: High-dose intravenous glutathione in man.: Pharmacokinetics and effects on cyst(e)ine levels in plasma and urine. Eur. J. Clin. Invest. 21: 103-110, 1991.
Hagen, T. M. and Jones, D. P. Role of glutathione transport in extrahepatic detoxication, in Glutathione Centennial: Molecular Perspectives and Clinical Implications, N. Taniguchi, T. Higashi, Y. Sakamoto and A. Meister, eds. Acad. Press, New York, 1990.
Jones, D. P., Hagen, T. M., Weber, R., Wierzbicka, G. T., and Bonkovsky, H. L.: Oral administration of glutathione (GSH) increases plasma GSH concentration in humans. FASEB J. 3: A1250 (5953), 1990.
Effects of Glutathione on the Circulatory System
Glutathione impacts many aspects of the circulatory system, including interactions with nitric oxide signaling, ischemia, and control over vascular endothelium.
Demopoulos, H. B., Flamm, E. S., Pietronigro, D. D., and Seligman, M. L.: Free radical pathology and antioxidants in regional cerebral ischemia and central nervous system trauma. In: Anesthesia and Neurosurgery, eds. J. E. Cottrell and H. Tunndorf, C. V. Mosby, St. Louis, 1986, pp. 246-279.
Kagan, V. E., Bakalova, R. A., Koynova, G. M., Tyurin, V. A., Seriniva, E. A., Petkov, V. V., Staneva, D. S. and Packer, L.: Antioxidant protection of the brain against oxidative stress. In: Free Radicals in the Brain, eds. L. Packer, L. Prilipko, and Y. Christen. Springer-Verlag, New York, 1992, pp. 49-61.
Pietronigro, D. D., Demopoulos, H. B., Hovsepian, M. and Flamm, E. S.: Brain ascorbic acid depletion during cerebral ischemia. Stroke 13: 117-119, 1982.
Shan, X., Aw, T. Y. and Jones, D. P.: Glutathione-dependent protection against oxidative injury. Pharmac. Ther. 47: 61-71, 1990.
Simon, D. I., Stamler, J. S., Jaraki, O., et al.: Antiplatelet properties of protein S-nitrosothiols derived from nitric oxide and endothelium-derived relaxing factor. Arterioscler. Thromb. 13 (6): 791-799, 1993.
Taccone-Gallucci, M., Lubrano, R., Clerico, A., Meloni, C., Morosetti, M., Meschini, L., Elli, M., Trapasso, E., Castello, M. A. & Casciani, C. U.: Administration of GSH has no influence on the RBC membrane: Oxidative damage to patients on hemodialysis. ASAIO Journal 38: 855-857, 1992.
Use of High-Dose Oral GSH in Cancer Patients.
In one published study, eight patients with hepatocellular carcinoma were treated with 5 g oral reduced glutathione per day. Two patients withdrew shortly after receiving GSH due to intolerable side-effects (gastrointestinal irritation and sulfur odor). The remaining patients, aged 27-63, three male and three female, did not experience side-effects from this high dose of GSH and continued to take 5 g oral GSH for periods ranging from 119 days (at which time the patient died from her tumor) to&gt;820 days (this patient was still alive at the time of publication and was still taking 5 g oral GSH daily; his tumor had not progressed and his general condition was good). Two of the female patients survived 1 year and exhibited regression or stagnation of their tumor growth. The remaining two patients, both male, died as expected within 6 months.
Experience in HIV-Infected Patients.
A commercially available nutritional formulation containing 3 grams of reduced glutathione was given daily to a group of 46 AIDS patients for a period of three months by a group of private physicians. No significant GSH-related adverse effects were reported. No evidence of toxicities from laboratory studies or from clinical examinations was reported; however, no benefit was conclusively demonstrated.
Pharmacokinetics of Glutathione
The pharmacokinetics of intravenously administered GSH were determined in the rat and interpreted by means of an open, two-compartment model. Following a bolus injection of 50-300 mmol/kg GSH, arterial plasma concentrations of (i) GSH, (ii) oxidized glutathione/GSSG, (iii) total thiols, and (iv) soluble thiols minus GSH, were elevated and then rapidly decreased non-exponentially, as anticipated. With increasing dose, the rate constant for drug elimination and plasma clearance increased form 0.84 to 2.44 mL/min. and the half-life of the elimination phase decreased from 52.4 to 11.4 minutes. Both the apparent volume of distribution and the degree of penetration of GSH into the tissues were diminished with increasing dose (from 3.78 to 1.33 L/Kg and from 6.0 to 0.51 as k.sub.12 /k.sub.21, respectively). The data indicate that GSH is rapidly eliminated. This is mainly due to rapid oxidation in plasma rather than by increased tissue extraction or volume distribution. Thus, plasma GSH levels appear to be quickly regulated by which the body may maintain concentrations within narrow physiological limits.
When single doses of 600 mg GSH were administered intravenously to sheep, GSH levels in venous plasma and lung lymph rose transiently. The mean concentration was approximately 50 mM for venous plasma, peaking at 30 min, and returning to baseline after 45 minutes. Lung lymph peak level was about 100 mM at 15 min, returning to baseline after 30 minutes. Average epithelial lining fluid (ELF) levels were variable but showed no significant increase over baseline during the three hour observation period. Urine excretion was rapid with peak levels at 15 minutes. In both plasma and lung lymph, GSH accounted for greater than 95% of the total glutathione (GSH plus GSSG). In ELF 75.4% of the baseline glutathione was in the reduced form, whereas in urine 59.6% was present as GSH.
Orally ingested reduced glutathione is absorbed intact from the small intestine in a rat model, specifically in the upper jejunum. It is noted that rat metabolism differs from man, and therefore the results of rat studies should be verified in man before the results are extrapolated. Plasma GSH concentration in rats increased from 15 to 30 mM after administration of GSH either as a liquid bolus (30 mM) or mixed (2.5-50 mg/g) in AIN-70 semi-synthetic diet (11). GSH concentration was maximal at 90-120 minutes after GSH administration and remained high for over 3 hours. Administration of the amino acid precursors of GSH had little or no effect on plasma GSH values, indicating that GSH catabolism and re-synthesis do not account for the increased GSH concentration seen. Inhibition of GSH synthesis and degradation by L-buthionine-[S,R]-sulfoximine (BSO) and acivicin showed that the increased plasma GSH came mostly from absorption of intact GSH instead of from its metabolism. Plasma protein-bound GSH also increased after GSH administration, with a time course similar to that observed for free plasma GSH. Thus, dietary GSH can be absorbed intact and results in a substantial increase in blood plasma GSH.
Administration of oral GSH increased hepatic glutathione levels in: (i) rats fasted 48 hours, (ii) mice treated with GSH depletors, and (iii) mice treated with paracetamol (a drug which promotes a depletion of hepatic GSH followed by hepatic centrilobular necrosis). In these experiments, the animals were orally intubated with 1000 mg/kg body weight GSH. Mean pretreatment values in 48-hour fasted rats were 3.0-3.1 mmol/g fresh hepatic tissue. Mean values after treatment were 5.8, 4.2, and 7.0 mmol/g fresh hepatic tissue for 2.5, 10, and 24 hours post-treatment, respectively. Mice were given an oral dose of GSH (100 mg/kg) and concentrations of GSH were measured at 30, 45 and 60 min in blood plasma and after 1 hr in liver, kidney, heart, lung, brain, small intestine and skin. GSH concentrations in plasma increased from 30 mM to 75 mM within 30 min of oral GSH administration, consistent with a rapid flux of GSH from the intestinal lumen to plasma. No increases over control values were obtained in most tissues except lung over the same time course. Mice pretreated with the GSH synthesis inhibitor BSO had substantially decreased tissue concentrations of GSH, and oral administration of GSH to these animals resulted in statistically-significant increases in the GSH concentrations of kidney, heart, lung, brain, small intestine and skin but not in liver.
Fahey, R. C., and Newton, G. L.: Determination of low molecular weight thiols using monobromobimane fluorescent labeling and high-performance liquid chromatography. Meth. Enzymol. 143: 85-96, 1987. See:
Mills, B. J., Richie, J. P. Jr., and Lang, C. A.: Sample processing alters glutathione and cysteine values in blood. Anal. Biochem. 184: 263-267, 1990.
Richie, J. P. Jr., and Lang, C. A.: The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection. Anal. Biochem. 163: 9-15, 1987.
Tietz, F.: Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues. Anal. Biochem. 27: 502-22, 1969.
The kinetics and the effect of glutathione on plasma and urine sulphydryls were studied in ten healthy human volunteers. Following the intravenous infusion of 2000 mg/m.sup.2 of GSH the concentration of total glutathione in plasma increased from 17.5-13.4 mmol/Liter (mean=/-SD) to 823-326 mmol/Liter. The volume of distribution of exogenous glutathione was 176-107 Ml/Kg and the elimination rate constant was 0.063-0.027/minute, corresponding to a half-life of 14.1-9.2 minutes. Cysteine in plasma increased from 8.9-3.5 mmol/Liter to 114-45 mmol/Liter after the infusion. In spite of the increase in cysteine, the plasma concentration of total cyst(e)ine (i.e. cysteine, cystine, and mixed disulphides) decreased, suggesting an increased uptake of cysteine from plasma into cells. The urinary excretion of glutathione and of cyst(e)ine was increased 300-fold and 10-fold respectively, in the 90 minutes following the infusion.
Normal healthy volunteers were given an oral dose of GSH to determine whether dietary GSH could raise plasma GSH levels. Results showed that an oral dose of GSH (15 mg/kg) raised plasma glutathione levels in humans 1.5-10 fold over the basal concentration in four out of five subjects tested, with a mean value three times that of normal plasma GSH levels. Plasma GSH became maximal 1 hour after oral administration, dropping to approximately 1/2 maximal values after three hours. Equivalent amounts of GSH amino acid constituents failed to increase plasma levels of GSH. GSH bound to plasma proteins also increased with the same time course as seen with free GSH.